Cancer associated glucose transporter 8 variant

ABSTRACT

The present invention provides the amino acid and nucleic acid sequences of heavy chain and light chain complementarity determining regions of a cancer specific antibody. In addition, the invention provides cancer specific antibodies and immunoconjugates comprising the cancer specific antibody attached to a toxin or label, and methods and uses thereof. The invention also relates to diagnostic methods and kits using the cancer specific antibodies of the invention. Further, the invention provides a novel cancer-associated antigen and its uses thereof.

INCORPORATION OF SEQUENCE LISTING

A computer readable form of the Sequence Listing“10241-109_SequenceListing.txt” (14,270 bytes), submitted via EFS-WEBand created on Feb. 15, 2011, is herein incorporated by reference.

FIELD OF THE INVENTION

The invention relates to human cancer-specific binding proteins and alluses thereof. In particular, the invention relates to antibodies orantibody fragments specific for antigens or molecules on cancer cellsand to immunoconjugates comprising the binding proteins of theinvention, and methods of use thereof. The invention also relates to anovel cancer associated-antigen and uses thereof.

BACKGROUND OF THE INVENTION

In the year 2000, an estimated 22 million people were suffering fromcancer worldwide and 6.2 millions deaths were attributed to this classof diseases. Every year, there are over 10 million new cases and thisestimate is expected to grow by 50% over the next 15 years (WHO, WorldCancer Report. Bernard W. Stewart and Paul Kleihues, eds. IARC Press,Lyon, 2003). Current cancer treatments are limited to invasive surgery,radiation therapy and chemotherapy, all of which cause eitherpotentially severe side-effects, non-specific toxicity and/ortraumatizing changes to ones body image and/or quality of life. Cancercan become refractory to chemotherapy reducing further treatment optionsand likelihood of success. The prognosis for some cancer is worse thanfor others and some are almost always fatal. In addition, some cancerswith a relatively high treatment success rate remain major killers dueto their high incidence rates.

One of the causes for the inadequacy of current cancer treatments istheir lack of selectivity for affected tissues and cells. Surgicalresection always involves the removal of apparently normal tissue as a“safety margin” which can increase morbidity and risk of complications.It also always removes some of the healthy tissue that may beinterspersed with tumor cells and that could potentially maintain orrestore the function of the affected organ or tissue. Radiation andchemotherapy will kill or damage many normal cells due to theirnon-specific mode of action. This can result in serious side-effectssuch as severe nausea, weight loss and reduced stamina, loss of hairetc., as well as increasing the risk of developing secondary cancerlater in life. Treatment with greater selectivity for cancer cells wouldleave normal cells unharmed thus improving outcome, side-effect profileand quality of life.

The selectivity of cancer treatment can be improved by using antibodiesthat are specific for molecules present only or mostly on cancer cells.Such antibodies can be used to modulate the immune system and enhancethe recognition and destruction of the cancer by the patient's ownimmune system. They can also block or alter the function of the targetmolecule and, thus, of the cancer cells. They can also be used to targetdrugs, genes, toxins or other medically relevant molecules to the cancercells. Such antibody-drug complexes are usually referred to asimmunotoxins or immunoconjugates and a number of such compounds havebeen tested in recent year [Kreitman R J (1999) Immunotoxins in cancertherapy. Curr Opin Immunol 11:570-578; Kreitman R J (2000) Immunotoxins.Expert Opin Pharmacother 1:1117-1129; Wahl R L (1994) Experimentalradioimmunotherapy. A brief overview. Cancer 73:989-992; Grossbard M L,Fidias P (1995) Prospects for immunotoxin therapy of non-Hodgkin'slymphoma. Clin Immunol Immunopathol 76:107-114; Jurcic J G, Caron P C,Scheinberg D A (1995) Monoclonal antibody therapy of leukemia andlymphoma. Adv Pharmacol 33:287-314; Lewis J P, DeNardo G L, DeNardo S J(1995) Radioimmunotherapy of lymphoma: a UC Davis experience. Hybridoma14:115-120; Uckun F M, Reaman G H (1995) Immunotoxins for treatment ofleukemia and lymphoma. Leuk Lymphoma 18:195-201; Kreitman R J, Wilson WH, Bergeron K, Raggio M, Stetler-Stevenson M, FitzGerald D J, Pastan I(2001) Efficacy of the anti-CD22 recombinant immunotoxin BL22 inchemotherapy-resistant hairy-cell leukemia. N Engl J Med 345:241-247].Most antibodies tested to date have been raised against known cancermarkers in the form of mouse monoclonal antibodies, sometimes“humanized” through molecular engineering. Unfortunately, their targetsare usually also present on subset of normal cells thus still causingsome non-specific effect. Furthermore, these antibodies are basicallymouse proteins that are being seen by the human patient's immune systemas foreign proteins. The ensuing immune reaction and antibody responsecan result in a loss of efficacy or in side-effects.

The inventors have used a different approach in their development ofantibodies for cancer treatment. Instead of immunizing experimentalanimals with cancer cells or isolated cancer cell markers, they havesought out only those markers that are recognized by the patient's ownimmune system or, in other words, that are seen by the immune system asa foreign molecule. This implies that the markers or antigens areusually substantially absent on normal cells and, thus, the risk ofnon-specific toxicity is further reduced. Hybridoma libraries aregenerated from cancer patient-derived lymphocytes and the antibodiesthey secrete are tested for binding to normal and tumor cells. Onlyantibodies showing high selectivity for cancer cells are retained forfurther evaluation and development as a cancer therapeutic or diagnosticagent. One such highly selective antibody is the subject of this patentapplication. In addition to being selective, this antibody is fullycompatible with the patient's immune system by virtue of being a fullyhuman protein. The antibody of the invention can be used for diagnosticor therapeutic uses or as a basis for engineering other bindingmolecules for the target antigen. The antibody of the invention can alsobe used to identify the target antigen. The antigen can then be used todesign new cancer treatment or diagnostics.

The basic structure of an antibody molecule consists of four proteinchains, two heavy chains and two light chains. These chains areinter-connected by disulfide bonds. Each light chain is comprised of alight chain variable region and a light chain constant region. Eachheavy chain is comprised of a heavy chain variable region and a heavychain constant region. The light chain and heavy chain variable regionscan be further subdivided into framework regions and regions ofhypervariability, termed complementarity determining regions (CDR). Eachlight chain and heavy chain variable region is composed of three CDRsand four framework regions.

Glucose transporter 8 (GLUT8) is a member of the GLUT family of proteinsand is known to have sugar transporting activity. GLUT8 is encoded bygene slc2a8, which is found on human chromosome 9. GLUT8 is 477 aminoacids in length. It is a ˜50 kDa type II transmembrane protein. It has12 transmembrane regions. It has a short extracellular loop between TM1and TM2 and a long extracellular loop between TM9 and TM10. Despitehaving several transmembrane regions, GLUT8 is located intracellularlylikely because of a N-terminal di-leucine motif (Ibberson et al. JBC275: 4607-4612, 2000; Moadel et al., Cancer Res 65:698-702, 2005).Translocation to the membrane has been observed in mouse cells uponinsulin treatment (Carayannopoulos et al., PNAS 97:7313-18, 2000) or inrat cells upon hypoxic shock or insulin treatment (U.S. Ser. No.09/886,954 [2002/0038464]). In human, membrane localization has not beenreported and no stimuli has been identified to induce translocation(Widmer et al., Endocrinology 146:4727-36, 2005).

GLUT/SLC2A family nomenclature has been published in: Amer. J. Physiol.Endocrinol. Metab. 282:E974-76, 2002. The name GLUT8 was used in thepast to describe what it now known as GLUT12—as indicated in that paper.The N-terminal di-leucine motif has been found in all mammalian GLUT8sequences (see Zhao et al., Biochimica et Biophysica Acta 1680:103-113,2004—showing bovine, human, rat, mouse).

SUMMARY OF THE INVENTION

The present inventors have prepared human cancer-specific antibodiesthat bind to several types of cancer cells, including breast cancer,ovarian cancer, prostate cancer, melanoma, liver cancer, colon cancer,cervical cancer, head & neck cancer, bladder cancer, stomach cancer,pancreatic cancer and endometrial cancer. Importantly, the antibodies donot significantly bind to normal tissue making them suitable candidatesfor cancer therapy and diagnosis.

The inventors have cloned and sequenced the antibodies and determinedthe sequence of the antibody light and heavy chain variable regions andcomplementarity determining regions 1, 2 and 3. Accordingly, theinvention provides isolated light chain complementarity determiningregions 1, 2 and/or 3, comprising the amino acid sequences RASQDISNYLA(SEQ ID NO:1), AASSLHS (SEQ ID NO:2) and LQYSTYPIT (SEQ ID NO:3),respectively; and isolated heavy chain complementarity determiningregions 1, 2 and 3, comprising the amino acid sequences NYAMS (SEQ IDNO:4), AITPSGGSTNYADSVKG (SEQ ID NO:5) and VPYRSTWYPLY (SEQ ID NO:6),respectively.

The invention also provides isolated nucleic acid sequences encodinglight chain complementarity determining regions 1, 2 and/or 3,comprising the amino acid sequences RASQDISNYLA (SEQ ID NO:1), AASSLHS(SEQ ID NO:2) and LQYSTYPIT (SEQ ID NO:3), respectively; and isolatednucleic acid sequences encoding heavy chain complementarity determiningregions 1, 2 and/or 3, comprising the amino acid sequences NYAMS (SEQ IDNO:4), AITPSGGSTNYADSVKG (SEQ ID NO:5) and VPYRSTWYPLY (SEQ ID NO:6),respectively.

Additional aspects of the invention are isolated light chain variableregions comprising light chain complementarity determining regions 1, 2and/or 3 of the invention (SEQ ID NOS:1-3), and isolated heavy chainvariable regions comprising heavy chain complementarity determiningregions 1, 2 and/or 3 of the invention (SEQ ID NOS:4-6). In oneembodiment, the light chain variable region comprises the amino acidsequence shown in FIG. 1 (SEQ ID NO:7). In another embodiment, the heavychain variable region comprises the amino acid sequence shown in FIG. 2(SEQ ID NO:9).

The invention also provides an isolated nucleic acid sequence encodingthe light chain variable region of the invention, and an isolatednucleic acid sequence encoding the heavy chain variable region of theinvention. In one embodiment, the light chain variable region comprisesthe nucleic acid sequence shown in FIG. 1 (SEQ ID NO: 8). In anotherembodiment, the heavy chain variable region comprises the nucleic acidsequence shown in FIG. 2 (SEQ ID NO:10).

Another aspect of the invention is a binding protein, preferably anantibody or antibody fragment, that comprises at least one light chaincomplementarity determining region of the invention (i.e. one or more ofthe SEQ ID NOS:1-3) and/or at least one heavy chain complementaritydetermining region of the invention (i.e. one or more of SEQ ID NO:4-6).The invention also provides a binding protein, preferably an antibody orantibody fragment that comprises the light chain variable regions of theinvention and/or the heavy chain variable regions of the invention.

The inventors have also identified the antigen to which the bindingproteins of the invention bind. Accordingly, the invention providesbinding proteins that bind to: glucose transporter 8 (GLUT8) or variantsthereof; a protein comprising any one of the amino acid sequences of SEQID NOS:11-20, preferably SEQ ID NOS:11-13; or a cancer-associatedvariant of GLUT8 that is expressed on the surface of cancer cells. Inone embodiment of the invention, the cancer-associated variant of GLUT8,comprises the amino acid sequence defined by any one of SEQ ID NOS: 11,12 or 13, or variants thereof. In another embodiment of the invention,the cancer-associated variant of GLUT8, comprises GLUT8 that has amodification in the N-terminal di-leucine motif. In a further embodimentof the invention, the N-terminal di-leucine motif has been modified todi-alanine.

In addition, the invention provides compositions comprising the bindingproteins of the invention, such as antibodies and antibody fragments,with a pharmaceutically acceptable excipient, carrier, buffer orstabilizer.

Another aspect of the invention is an immunoconjugate comprising (1)binding protein of the invention, preferably an antibody or antibodyfragment that binds to an antigen or molecule on a cancer cell, attachedto (2) an effector molecule. A further aspect of the invention is animmunoconjugate comprising (1) binding protein of the invention,preferably an antibody or antibody fragment that binds to an antigen ormolecule that is internalized by a cancer cell, attached to (2) aneffector molecule. In a preferred embodiment, the effector molecule is(i) a label, which can generate a detectable signal, directly orindirectly, or (ii) a cancer therapeutic agent, which is eithercytotoxic, cytostatic or otherwise prevents or reduces the ability ofthe cancer cells to divide and/or metastasize. Preferably, the cancertherapeutic agent is a toxin or cytotoxin.

The invention also provides compositions comprising the immunoconjugateof the invention and uses of the immunoconjugate for the manufacture ofa medicament for treating or preventing cancer, and diagnostic purposes.In addition, the invention provides methods of treating or preventingcancer using the immunoconjugate of the invention and related kits.

A further aspect of the invention is a method of detecting or monitoringcancer in a subject comprising the steps of:

-   -   (1) contacting a test sample taken from said subject with a        binding protein of the invention and that binds specifically to        an antigen on the cancer cell to produce a binding        protein-antigen complex;    -   (2) measuring the amount of binding protein-antigen complex in        the test sample; and    -   (3) comparing the amount of binding protein-antigen complex in        the test sample to a control.

Another aspect of the invention is a diagnostic agent comprising theimmunoconjugate of the invention, wherein the effector molecule is alabel, which can generate a detectable signal, directly or indirectly.

The invention also includes an isolated protein that can specificallybind with one of the binding proteins of the invention, nucleic acidsequences and uses thereof.

The inventors have identified the antigen to which the binding proteinsof the invention bind. The invention includes the novel-cancerassociated antigen, which is a variant of GLUT8 that is expressed on thesurface of cancer cells. The invention also includes the use of thenovel cancer-associated antigen of the invention in the treatment anddiagnosis of cancer.

In an embodiment of the invention, the cancer-associated variant ofGLUT8, comprises the amino acid sequence defined by any one of SEQ IDNOS: 11, 12 or 13, or variants thereof. In another embodiment of theinvention, the cancer-associated variant of GLUT8, comprises GLUT8 thathas a modification in the N-terminal di-leucine motif. In a furtherembodiment of the invention, the N-terminal di-leucine motif has beenmodified to di-alanine.

The invention also includes methods of detecting or monitoring cancer ina subject having or suspected of having cancer, comprising detecting acancer-associated variant of GLUT8 on a cell in the sample, whereincancer is indicated, if the cancer-associated variant of GLUT8 isdetected on the cell.

In addition, the invention includes methods of detecting or monitoringcancer in a subject having or suspected of having cancer, comprisingdetecting the expression of a cancer-associated variant of GLUT8 in thecell in the sample, wherein cancer is indicated, if the expression ofthe cancer-associated variant of GLUT8 is detected in the cell.

A further aspect of the invention is a method of treating or preventingcancer in a subject by modulating the function or expression of GLUT8 inthe cancer cell.

The invention also includes methods of treating or preventing cancer ina subject using the cancer-associated variant of GLUT8 or fragmentsthereof. In addition, the invention includes pharmaceutical compositionscomprising an effective amount a cancer-associated variant of GLUT8 orfragments thereof, nucleic acid sequences encoding the cancer-associatedvariant of GLUT8 or fragments thereof, and/or recombinant expressionvectors comprising nucleic acid sequences encoding the cancer-associatedvariant of GLUT8 or fragments thereof.

Other features and advantages of the present invention will becomeapparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples while indicating preferred embodiments of the invention aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will now be described in relation to the drawings inwhich:

FIG. 1 is the nucleic acid (SEQ ID NO:8) and amino acid (SEQ ID NO:7)sequence of the light chain variable region of VB1-050.

FIG. 2 is the nucleic acid (SEQ ID NO:10) and amino acid (SEQ ID NO:9)sequence of the heavy chain variable region of VB1-050.

FIG. 3 demonstrates antibody cell surface binding after incubation ofA-375 cells at different temperatures as determined by flow cytometry.Fluorescence labeling of A-375 cells after incubation of cellsuspensions at 4° C.: 4B5 (1) and VB1-050 (2). Fluorescence labeling ofA-375 cells after warming antibody-bound cells to 37° C.: VB1-050 for 60min (3), for 120 min (4).

FIG. 4 shows confocal microscopy assessment of VB1-050 internalization.A-375 cells were incubated with antibody at 4° C., washed and warmed to37° C. for 60 min. Cells were fixed, permeabilized and labeled withfluorescent-labeled second antibody. Fluorescence labeling of A-375cells after incubation of VB1-050 at 4° C. for 60 min, displayingcircumferential surface distribution of labeling, (60××4) magnification(A). Following incubation of antibody-bound cells at 37° C. for 60 minthe cells show strong intracellular staining by internalized antibody,(60××4) magnification (B).

FIG. 5 shows an agarose gel of the PCR reaction. The DNA was detectedusing ethidium bromide under a UV lamp. A) ThePelB—V_(H845)—C_(H)—F-de-bouganin/psV73 plasmid andPelB(—S)—V_(H050)—C_(H)—F-de-bouganin/pSV73 were digested with EcoRI andPvuII and loaded on lane 1 and 3, respectively. The * symbol indicatesthe EcoRI-PelB-PvuII insert with the peptide leader sequence which wasligated to the PelB(—S)—V_(H050)—C_(H)—F-de-bouganin/pSV73 pre-digested(indicated with the arrow) to create thePelB—V_(H050)—C_(H)—F-de-bouganin/pSV73. B) ThePelB—(S)—V_(L050)—C_(L)/pSV73 andSpeI-de-bouganin-PelB—V_(L845)—C_(L)/pSV73 plasmid were digested withEcoRV and XhoI and loaded on lanes 2-3 and 4-5, respectively. The insertand the vector indicate with the * symbol and the arrow, respectivelywere used to create the SpeI-de-bouganin-PelB—V_(L845)—C_(L)/pSV73plasmid which was subsequently inserted into the 3302 plasmid. C) TheSpeI-de-bouganin-PelB—V_(L845)—C_(L)/3302 plasmid and D)PelB—V_(H050)—C_(H)—F-de-bouganin insert, digested with EcoRI and SpeI(indicated with the arrow and the * symbol, respectively) and loaded onlane 2 were ligated to create VB6-050/3302.

FIG. 6 shows a Western blot of VB6-050. A representative supernatant ofVB6-845 (lane 1) and VB6-050 (lane 2) was loaded under non-reducingconditions on a SDS-PAGE gel and immunoblotted with an anti-human Kappalight chain-HRP antibody ( 1/1000). Lanes 3 and 4 correspond to thesupernatant of a non-induced culture and the ladder, respectively. Lane5 is VB6-845 supernatant previously tested positive on Western blot.

FIG. 7 shows a Western blot of a representative purification of VB6-050from E104 supernatant. A) Samples, 16 μL, taken at different steps ofthe purification process were immunoblotted using anti-human Kappalight-HRP antibody. The arrows indicate intact product. Lane 1: Culturesupernatant; lane 2: Permeate of the concentrated supernatant; lane 3:Concentrated supernatant 1/10 diluted; lane 4: Permeate of thediafiltered concentrated supernatant; lane 5: Diafiltered concentratedsupernatant 1/10; lane 6: Flow-through of the CM-sepharose column; lane7: Wash of the CM-sepharose column; lane 8: Eluate of the CM-sepaharosecolumn or Ni-sepharose starting material; lane 9: Flow-through of theNi-chelating column; lanes 10, 11 and 12: Different step washes of theNi-chelating column; lane 13: Eluate of the Ni-chelating-Sepharose orSEC-200 starting material; lane 14: Pool of the SEC-200 fractions 26-28;lane 15: Ladder and VB6-845 as a control. B) Coomassie staining ofVB6-050. Lane 1: SEC-200 starting material; lane 2: fraction 27; lane 3:purified VB6-845; lane 4: ladder.

FIG. 8 shows the titration curve of VB6-050. SKBR-3, A-375 and SK-OV-3cells were incubated with various concentrations of VB6-050 and themedium fluorescence was obtained by flow cytometry. The MedianFluorescence (MF) Fold Increase was calculated using the followingformula, MF Fold increase=MF measured at each concentration/MF measuredwith PBS.

FIG. 9 shows the in vitro cytotoxicity of VB6-050. MTS assay of VB6-050with antigen-positive cells MB-435S (open circle) and antigen-negativecells Daudi (black circle). Cells seeded at 1000 cells per well, wereincubated with the Fab-de-bouganin purified proteins. After 5 daysincubation, the cell viability was measured and IC₅₀ was determined.

FIG. 10 shows the fractionation profiles of HepG2, MCF-7, Panc-1 andC-33A on a PF-2D system. A comparative profile of the differences inantigen expression between two positive and two negative cell lines.This figure represents a chromatographic file from 10 to 25 minutes. Aclear view of the separated antigen differences is visualized in bothpositive cell lines. MCF-7 and HepG2 showed two peaks eluting at 15 and18 minutes, indicating moderate levels of hydrophobicity. Panc-1 andC-33A showed no corresponding peaks. A peak at 12 minutes was observedin all cell lines.

FIG. 11 shows the TOF-MS scans of peptides obtained from HepG2 cellline, to detect the presence of all peptide ions in the sample.Fifty-three scans at 1200-1400V in the range of 100-1200 amu on a staticnanospray resulted in the recovery of a significant number of peptides,which when analyzed yielded a protein ID as Glucose Transporter 8.

FIG. 12 shows the TOF-MS scans of peptides obtained from Panc-1 cellline, to detect the presence of all peptide ions in the sample. Thirtyscans at 1200-1400V in the range of 100-1200 amu on a static nanosprayresulted in the recovery of a significant number of peptides, which whenanalyzed yielded a protein ID as IgG.

FIG. 13 shows the TOF-MS scans of peptides obtained from MCF-7 cellline, to detect the presence of all peptide ions in the sample.Twenty-seven scans at 1200-1400V in the range of 100-1200 amu on astatic nanospray resulted in the recovery of a significant number ofpeptides, which when analyzed yielded a protein ID as GlucoseTransporter 8.

FIG. 14 shows the TOF-MS scans of peptides obtained from C-33A cellline, to detect the presence of all peptide ions in the sample. Thirtyscans at 1200-1400V in the range of 100-1200 amu on a static nanosprayresulted in the recovery of a significant number of peptides, which whenanalyzed yielded a protein ID as IgG.

FIG. 15 shows the sequence coverage of peptides recovered from massspectrometry analysis as listed in Table 8. A total of 8 peptides wererecovered from in-solution tryptic digestion and 34% coverage of theprotein was obtained. Sequences underlined represent the peptidesequences recovered and bolded sequences show the variant amino acidsequences.

FIG. 16 shows the peptide mass fingerprinting results for the peptidesrecovered from VB1-050Ag. Protein scores greater than 64 were consideredsignificant. The only significant protein IDs observed pointed to theone antigen, known as Glucose Transporter 8.

FIG. 17 shows that the identified antigen, glucose transporter 8, has asignificant score of 83. Due to the nature of the database server andthe similarity/homology linked proteins, all the isoforms of thisprotein were pulled down as hits. MS/MS fragmentation and identity ofpeptides confirms that the antigen is glucose transporter 8.

FIG. 18 shows the MS/MS ion fragmentation of the neutral peptide Mr.1401.54, appearing as a triply charged molecule (466.60000, 3+). Thepeptide sequence (SEQ ID NO:19) exactly matched the peptide from GlucoseTransporter 8.

FIG. 19 shows the MS/MS ion fragmentation of the neutral peptide Mr.1070.785, appearing as a doubly charged molecule (536.40000, 2+). Thepeptide sequence (SEQ ID NO:20) exactly matched the peptide from GlucoseTransporter 8.

FIG. 20 shows the MS/MS ion fragmentation of the neutral peptide Mr.1997.9992, appearing as a triply charged molecule (667.098230, 3+). Thepeptide sequence (SEQ ID NO:12) showed changes in amino acids atpositions 7, 10, 12, 13, 14, 15 and 18; compared to the homologouspeptide from Glucose Transporter 8.

FIG. 21 shows the MS/MS ion fragmentation of the neutral peptide Mr.1176.3547, appearing as a doubly charged molecule (589.100000, 2+). Thepeptide sequence (SEQ ID NO:30) showed changes in amino acids atpositions 7, 10, 12, 13, 14 and 15; compared to the homologous peptidefrom Glucose Transporter 8.

DETAILED DESCRIPTION OF THE INVENTION (A) Definitions

The term “administered systemically” as used herein means that theimmunoconjugate and/or other cancer therapeutic may be administeredsystemically in a convenient manner such as by injection (subcutaneous,intravenous, intramuscular, etc.), oral administration, inhalation,transdermal administration or topical application (such as topical creamor ointment, etc.), suppository applications, or means of an implant. Animplant can be of a porous, non-porous, or gelatinous material,including membranes, such as sialastic membranes, or fibers.Suppositories generally contain active ingredients in the range of 0.5%to 10% by weight.

The term “amino acid” includes all of the naturally occurring aminoacids as well as modified amino acids.

The term “antibody” as used herein is intended to include monoclonalantibodies, polyclonal antibodies, and chimeric antibodies. The antibodymay be from recombinant sources and/or produced in transgenic animals.The term “antibody fragment” as used herein is intended to include Fab,Fab′, F(ab′)₂, scFv, dsFv, ds-scFv, dimers, minibodies, diabodies, andmultimers thereof and bispecific antibody fragments. Antibodies can befragmented using conventional techniques. For example, F(ab′)₂ fragmentscan be generated by treating the antibody with pepsin. The resultingF(ab′)₂ fragment can be treated to reduce disulfide bridges to produceFab′ fragments. Papain digestion can lead to the formation of Fabfragments. Fab, Fab′ and F(ab′)₂, scFv, dsFv, ds-scFv, dimers,minibodies, diabodies, bispecific antibody fragments and other fragmentscan also be synthesized by recombinant techniques.

The term “antibody or antibody fragment of the invention” as used hereincomprises at least one light chain complementarity determining region ofthe invention (i.e. one or more of SEQ ID NOS:1-3) and/or at least oneheavy chain complementarity determining region of the invention (i.e.one or more of SEQ ID NOS:4-6). Preferably, the antibody or antibodyfragment comprises the light chain CDR sequences (SEQ ID NOS:1-3) and/orthe heavy chain CDR sequences (SEQ ID NOS:4-6) or functional variants ofthe sequences so that the antibody or antibody fragment can bind to thecancer cell without substantially binding to normal cells. Antibodies orantibody fragments of the invention also include antibodies or antibodyfragments that bind to glucose transporter 8 (GLUT8) or variantsthereof, or a protein comprising any one of the amino acid sequences ofSEQ ID NOS: 11-20, preferably SEQ ID NOS:11-13.

By “at least moderately stringent hybridization conditions” it is meantthat conditions are selected which promote selective hybridizationbetween two complementary nucleic acid molecules in solution.Hybridization may occur to all or a portion of a nucleic acid sequencemolecule. The hybridizing portion is typically at least 15 (e.g. 20, 25,30, 40 or 50) nucleotides in length. Those skilled in the art willrecognize that the stability of a nucleic acid duplex, or hybrids, isdetermined by the Tm, which in sodium containing buffers is a functionof the sodium ion concentration and temperature (Tm=81.5° C.-16.6 (Log10 [Na+])+0.41(%(G+C)−600/l), or similar equation). Accordingly, theparameters in the wash conditions that determine hybrid stability aresodium ion concentration and temperature. In order to identify moleculesthat are similar, but not identical, to a known nucleic acid molecule a1% mismatch may be assumed to result in about a 1° C. decrease in Tm,for example if nucleic acid molecules are sought that have a >95%identity, the final wash temperature will be reduced by about 5° C.Based on these considerations those skilled in the art will be able toreadily select appropriate hybridization conditions. In preferredembodiments, stringent hybridization conditions are selected. By way ofexample the following conditions may be employed to achieve stringenthybridization: hybridization at 5× sodium chloride/sodium citrate(SSC)/5×Denhardt's solution/1.0% SDS at Tm−5° C. based on the aboveequation, followed by a wash of 0.2×SSC/0.1% SDS at 60° C. Moderatelystringent hybridization conditions include a washing step in 3×SSC at42° C. It is understood, however, that equivalent stringencies may beachieved using alternative buffers, salts and temperatures. Additionalguidance regarding hybridization conditions may be found in: CurrentProtocols in Molecular Biology, John Wiley & Sons, N.Y., 2002, and in:Sambrook et al., Molecular Cloning: a Laboratory Manual, Cold SpringHarbor Laboratory Press, 2001.

The term “binding protein” as used herein refers to proteins thatspecifically bind to another substance. In an embodiment, bindingproteins are antibodies or antibody fragments.

The term “binding proteins of the invention” as used herein includesantibodies or antibody fragments of the invention.

By “biologically compatible form suitable for administration in vivo” ismeant a form of the substance to be administered in which any toxiceffects are outweighed by the therapeutic effects.

The term “cancer” as used herein includes any cancer that can be boundby a binding protein of the invention, preferably an antibody orantibody fragment of the invention.

The term “cancer-associated variant of glucose transporter 8” as usedherein refers to a novel variant of glucose transporter 8 that isexpressed on the surface of cancer cells. In one embodiment of theinvention a cancer-associated variant of GLUT8 has the same function asGLUT8 as a transporter of sugar, but a different localization in thecell. For example, the cancer-associated variant of GLUT8 as the samefunction as GLUT8, as a transporter of sugar, but is localized to thesurface of the cell. In another embodiment, the cancer-associatedvariant of glucose transporter 8 is a protein comprising the amino acidsequence defined by SEQ ID NO: 11. In an additional embodiment, thecancer-associated variant of glucose transporter 8 is a proteincomprising the amino acid sequence defined by SEQ ID NO:12. In a furtherembodiment, the cancer-associated variant of glucose transporter 8 is aprotein comprising the amino acid sequence defined by SEQ ID NO:13. Inanother embodiment of the invention, the cancer-associated variant ofGLUT8, comprises GLUT8 that has a modification in the N-terminaldi-leucine motif. In a further embodiment of the invention, theN-terminal di-leucine motif has been modified to di-alanine.

A “conservative amino acid substitution”, as used herein, is one inwhich one amino acid residue is replaced with another amino acid residuewithout abolishing the protein's desired properties.

A control can be used in the method. The term “control” as used hereinrefers to a sample from a subject or a group of subjects who are eitherknown as having cancer or not having cancer.

The term “controlled release system” as used means the immunoconjugateand/or other cancer therapeutic of the invention can be administered ina controlled fashion. For example, a micropump may deliver controlleddoses directly into the area of the tumor, thereby finely regulating thetiming and concentration of the pharmaceutical composition (see, e.g.,Goodson, 1984, in Medical Applications of Controlled Release, vol. 2,pp. 115-138).

The term “derivative of a peptide” refers to a peptide having one ormore residues chemically derivatized by reaction of a functional sidegroup. Such derivatized molecules include for example, those moleculesin which free amino groups have been derivatized to form aminehydrochlorides, p-toluene sulfonyl groups, carbobenzoxy groups,t-butyloxycarbonyl groups, chloroacetyl groups or formyl groups. Freecarboxyl groups may be derivatized to form salts, methyl and ethylesters or other types of esters or hydrazides. Free hydroxyl groups maybe derivatized to form O-acyl or O-alkyl derivatives. The imidazolenitrogen of histidine may be derivatized to form N-im-benzylhistidine.Also included as derivatives are those peptides which contain one ormore naturally occurring amino acid derivatives of the twenty standardamino acids. For examples: 4-hydroxyproline may be substituted forproline; 5-hydroxylysine may be substituted for lysine;3-methylhistidine may be substituted for histidine; homoserine may besubstituted for serine; and ornithine may be substituted for lysine.

The phrase “detecting or monitoring cancer” refers to a method orprocess of determining if a subject has or does not have cancer or theextent of cancer. In addition, the binding proteins of the invention canbe used to detect or monitor the appearance and progression of thedisease.

The term “direct administration” as used herein means theimmunoconjugate and/or other cancer therapeutic may be administered,without limitation, intratumorally, intravascularly, and peritumorally.For example, the immunoconjugate may be administered by one or moredirect injections into the tumor, by continuous or discontinuousperfusion into the tumor, by introduction of a reservoir of theimmunoconjugate, by introduction of a slow-release apparatus into thetumor, by introduction of a slow-release formulation into the tumor,and/or by direct application onto the tumor. By the mode ofadministration “into the tumor,” introduction of the immunoconjugateand/or other cancer therapeutic to the area of the tumor, or into ablood vessel or lymphatic vessel that substantially directly flows intothe area of the tumor, is included.

As used herein, the phrase “effective amount” means an amount effective,at dosages and for periods of time necessary to achieve the desiredresult. Effective amounts of an immunoconjugate may vary according tofactors such as the disease state, age, sex, weight of the animal.Dosage regime may be adjusted to provide the optimum therapeuticresponse. For example, several divided doses may be administered dailyor the dose may be proportionally reduced as indicated by the exigenciesof the therapeutic situation.

“Glucose transporter 8” (GLUT8) is a protein encoded by gene slc2a8,which is found on human chromosome 9. It is a member of class III of theGLUT family of proteins and is known to have sugar transportingactivity. GLUT8 is 477 amino acids in length. It is a ˜50 kDa type IItransmembrane protein. It has 12 transmembrane regions. It has a shortputative extracellular loop between TM1 and TM2 and a long extracellularloop between TM9 and TM10. The term includes variants of GLUT8.(GLUT/SLC2A family nomenclature: Amer. J. Physiol. Endocrinol. Metab.282:E974-76, 2002.)

The term “heavy chain complementarity determining region” as used hereinrefers to regions of hypervariability within the heavy chain variableregion of an antibody molecule. The heavy chain variable region hasthree complementarity determining regions termed heavy chaincomplementarity determining region 1, heavy chain complementaritydetermining region 2 and heavy chain complementarity determining region3 from the amino terminus to carboxy terminus.

The term “heavy chain variable region” as used herein refers to thevariable region of a heavy chain.

The term “immunoconjugate of the invention” is used herein comprises (1)a binding protein, preferably an antibody or antibody fragment, of theinvention attached to (2) an effector molecule. The effector moleculecan be any molecule that one wishes to deliver to the cancer cell,including, but not limited to (i) a label, which can generate adetectable signal, directly or indirectly, or (ii) a cancer therapeuticagent, such as a toxin that is either cytotoxic, cytostatic or otherwiseprevents or reduces the ability of the cancer cells to divide and/ormetastasize.

The term “isolated nucleic acid sequences” as used herein refers to anucleic acid substantially free of cellular material or culture mediumwhen produced by recombinant DNA techniques, or chemical precursors, orother chemicals when chemically synthesized. An isolated nucleic acid isalso substantially free of sequences which naturally flank the nucleicacid (i.e. sequences located at the 5′ and 3′ ends of the nucleic acid)from which the nucleic acid is derived. The term “nucleic acid” isintended to include DNA and RNA and can be either double stranded orsingle stranded.

The term “isolated proteins” refers to a protein substantially free ofcellular material or culture medium when produced by recombinant DNAtechniques, or chemical precursors or other chemicals when chemicallysynthesized. It includes the light chain complementarity regions 1, 2and 3 of the invention, heavy chain complementarity regions 1, 2 and 3of the invention, light chain variable regions of the invention, heavychain variable regions of the invention, binding proteins of theinvention and antigen to which the binding proteins of the inventionbind.

The term “light chain complementarity determining region” as used hereinrefers to regions of hypervariability within the light chain variableregion of an antibody molecule. Light chain variable regions have threecomplementarity determining regions termed light chain complementaritydetermining region 1, light chain complementarity determining region 2and light chain complementarity determining region 3 from the aminoterminus to the carboxy terminus.

The term “light chain variable region” as used herein refers to thevariable region of a light chain.

The phrase “modification in the N-terminal di-leucine motif” in GLUT8refers to a change in the N-terminal di-leucine motif which effects thelocalization of GLUT8 so that GLUT8 is expressed on the surface of thecell, preferably a cancer cell. In one embodiment of the invention, theN-terminal di-leucine motif is changed to a di-alanine.

The term “modified bouganin” as used here means a modified bouganin thathas a reduced propensity to activate an immune response as described inPCT/CA2005/000410 and U.S. patent application Ser. No. 11/084,080. Inone example, the modified bouganin has the amino acid sequence (SEQ IDNO: 29):

YNTVSFNLGEAYEYPTFIQDLRNELAKGTPVCQLPVTLQTIADDKRFVLVDITTTSKKTVKVAIDVTDVYVVGYQDKWDGKDRAVFLDKVPTVATSKLFPGVTNRVTLTFDGSYQKLVNAAKADRKALELGVNKLEFSIEAIHGKTINGQEAAKFFLIVIQMVSEAARFKYIETEVVDRGLYGSFKPNFKVLNLENNWGDISDAIHKSSPQCTTINPALQLISPSNDPWVVNKVSQISPDMGILKFKSS K.

The term “nucleic acid sequence” as used herein refers to a sequence ofnucleoside or nucleotide monomers consisting of naturally occurringbases, sugars and intersugar (backbone) linkages. The term also includesmodified or substituted sequences comprising non-naturally occurringmonomers or portions thereof. The nucleic acid sequences of the presentinvention may be deoxyribonucleic acid sequences (DNA) or ribonucleicacid sequences (RNA) and may include naturally occurring bases includingadenine, guanine, cytosine, thymidine and uracil. The sequences may alsocontain modified bases. Examples of such modified bases include aza anddeaza adenine, guanine, cytosine, thymidine and uracil; and xanthine andhypoxanthine.

The term “sample” as used herein refers to any fluid, cell or tissuesample from a subject which can be assayed for cancer.

The term “sequence identity” as used herein refers to the percentage ofsequence identity between two polypeptide sequences. In order todetermine the percentage of identity between two polypeptide sequences,the amino acid sequences of such two sequences are aligned, preferablyusing the Clustal W algorithm (Thompson, J D, Higgins D G, Gibson T J,1994, Nucleic Acids Res. 22 (22): 4673-4680), together with BLOSUM 62scoring matrix (Henikoff S, and Henikoff J. G., 1992, Proc. Natl. Acad.Sci. USA 89: 10915-10919) and a gap opening penalty of 10 and gapextension penalty of 0.1, so that the highest order match is obtainedbetween two sequences wherein at least 50% of the total length of one ofthe sequences is involved in the alignment. Other methods that may beused to align sequences are the alignment method of Needleman and Wunsch(J. Mol. Biol., 1970, 48: 443), as revised by Smith and Waterman (Adv.Appl. Math., 1981, 2: 482) so that the highest order match is obtainedbetween the two sequences and the number of identical amino acids isdetermined between the two sequences. Other methods to calculate thepercentage identity between two amino acid sequences are generally artrecognized and include, for example, those described by Carillo andLipton (SIAM J. Applied Math., 1988, 48:1073) and those described inComputational Molecular Biology, Lesk, e.d. Oxford University Press, NewYork, 1988, Biocomputing: Informatics and Genomics Projects. Generally,computer programs will be employed for such calculations. Computerprograms that may be used in this regard include, but are not limitedto, GCG (Devereux et al., Nucleic Acids Res., 1984, 12: 387) BLASTP,BLASTN and FASTA (Altschul et al., J. Molec. Biol., 1990: 215: 403).

The phrase “N-terminal di-leucine motif” refers to the N-terminaldi-leucine motif in GLUT8 that is involved in localization of theprotein to the intracellular compartment of the cell. In one embodimentof the invention, the di-leucine motif is at positions 12 to 13 ofGLUT8.

The term “subject” as used herein refers to any member of the animalkingdom, preferably a mammal, more preferably a human being. In apreferred embodiment, the subject is suspected of having or has cancer.

As used herein, the phrase “treating cancer” refers to inhibiting cancercell replication, inhibiting cancer spread (metastasis), inhibitingtumor growth, reducing cancer cell number or tumor growth, decreasingthe malignant grade of a cancer (e.g., increased differentiation), orimproving cancer-related symptoms.

The term “variant” as used herein includes modifications or chemicalequivalents of the amino acid and nucleotide sequences of the presentinvention that perform substantially the same function as the proteinsor nucleic acid molecules of the invention in substantially the sameway. For example, variants of proteins of the invention include, withoutlimitation, conservative amino acid substitutions. Variants of proteinsof the invention also include additions and deletions to the proteins ofthe invention. In addition, variant peptides and variant nucleotidesequences include analogs and derivatives thereof.

(B) Proteins and Nucleic Acids of the Invention

(i) Light and Heavy Chain Complementarity Determining Regions and Lightand Heavy Chain Variable Regions

The invention provides isolated light chain complementarity determiningregion 1 comprising the amino acid sequence RASQDISNYLA (SEQ ID NO:1).The invention also provides isolated light chain complementaritydetermining region 2 comprising the amino acid sequence AASSLHS (SEQ IDNO:2). In addition, the invention provides isolated light chaincomplementarity determining region 3 comprising the amino acid sequenceLQYSTYPIT (SEQ ID NO:3).

The invention provides isolated heavy chain complementarity determiningregion 1 comprising the amino acid sequence NYAMS (SEQ ID NO:4). Theinvention also provides isolated heavy chain complementarity determiningregion 2 comprising the amino acid sequence AITPSGGSTNYADSVKG (SEQ IDNO:5). In addition, the invention provides isolated heavy chaincomplementarity determining region 3 comprising the amino acid sequenceVPYRSTWYPLY (SEQ ID NO:6).

The invention provides isolated light chain complementarity determiningregions 1, 2 and 3, comprising the amino acid sequences RASQDISNYLA (SEQID NO:1), AASSLHS (SEQ ID NO:2) and LQYSTYPIT (SEQ ID NO:3),respectively; and isolated heavy chain complementarity determiningregions 1, 2 and 3, comprising the amino acid sequences NYAMS (SEQ IDNO:4), AITPSGGSTNYADSVKG (SEQ ID NO:5) and VPYRSTWYPLY (SEQ ID NO:6),respectively.

The invention also includes variants of the CDR sequences that can bindto the same epitope or antigen recognized by the CDR sequences disclosedabove.

Additional aspects of the invention are isolated light chain variableregions comprising light chain complementarity determining regions 1, 2and/or 3 of the invention (SEQ ID NOS:1-3); and heavy chain variableregions comprising the heavy chain complementarity determining regions1, 2 and/or 3 of the invention (SEQ ID NOS:4-6). In one embodiment, thelight chain variable region comprises the amino acid sequence shown inFIG. 1 (SEQ ID NO:7), and the heavy chain variable region comprises theamino acid sequence shown in FIG. 2 (SEQ ID NO:9).

The invention also includes variants of the isolated light chainvariable regions and heavy chain variable regions that can bind to thesame epitope or antigen recognized by the isolated light chain variableregions and isolated heavy chain variable regions disclosed above.

A person skilled in the art will appreciate that the invention includesvariants to the amino acid sequences of SEQ ID NOS:1-6, 7 and 9,including chemical equivalents to the sequences disclosed by the presentinvention. Such equivalents include proteins that perform substantiallythe same function as the specific proteins disclosed herein insubstantially the same way. A functional variant of a CDR sequence willbe able to bind to the antigen or epitope recognized by the native CDRsequence. For example, equivalents include, without limitation,conservative amino acid substitutions.

In one embodiment, the variant amino acid sequences of the light chaincomplementarity determining regions 1, 2 and 3, and the heavy chaincomplementarity determining regions 1, 2 and 3 have at least 50%,preferably at least 60%, more preferably at least 70%, most preferablyat least 80%, and even more preferably at least 90% sequence identity toSEQ ID NOS:1-6, respectively.

In another embodiment, the variant amino acid sequences of the lightchain variable region and the heavy chain variable region have at least50%, preferably at least 60%, more preferably at least 70%, mostpreferably at least 80%, and even more preferably at least 90% sequenceidentity to SEQ ID NOS:7 and 9, respectively.

The invention also provides an isolated nucleic acid sequence encodingthe light chain variable region of the invention, and an isolatednucleic acid sequence encoding the heavy chain variable region of theinvention. In one embodiment, the light chain variable region comprisesthe nucleic acid sequence shown in FIG. 1 (SEQ ID NO: 8). In anotherembodiment, the heavy chain variable region comprises the nucleic acidsequence shown in FIG. 2 (SEQ ID NO:10). The invention also includesvariants to the nucleic acid sequences that encode for the light chainvariable region and heavy chain variable region of the invention. Forexample, the variants include nucleotide sequences that hybridize to thenucleic acid sequences encoding the light chain variable region andheavy chain variable region of the invention under at least moderatelystringent hybridization conditions.

The invention also provides isolated nucleic acid sequences encodinglight chain complementarity determining regions 1, 2 and/or 3,comprising the amino acid sequences RASQDISNYLA (SEQ ID NO:1), AASSLHS(SEQ ID NO:2) and LQYSTYPIT (SEQ ID NO:3), respectively; and isolatednucleic acid sequences encoding heavy chain complementarity determiningregions 1, 2 and/or 3, comprising the amino acid sequences NYAMS (SEQ IDNO:4), AITPSGGSTNYADSVKG (SEQ ID NO:5) and VPYRSTWYPLY (SEQ ID NO:6),respectively. The invention also provides an isolated nucleic acidsequence encoding the light chain variable region shown in FIG. 1 (SEQID NO:7), and an isolated nucleic acid sequence encoding the heavy chainvariable region shown in FIG. 2 (SEQ ID NO:9).

The invention also includes isolated nucleic acid sequences encodingvariants of the CDR sequences and variable region sequences discussedabove.

Variant nucleic acid sequences include nucleic acid sequences thathybridize to the nucleic acid sequences encoding the amino acidsequences shown in SEQ ID NOS:1-6, 7 and 9 and variants thereof under atleast moderately stringent hybridization conditions.

(ii) Binding Proteins

Another aspect of the invention is a binding protein, preferably anantibody or antibody fragment, that comprises at least one light chaincomplementarity determining region of the invention (i.e. one or more ofSEQ ID NOS:1-3) and/or at least one heavy chain complementaritydetermining region of the invention (i.e. one or more of SEQ IDNOS:4-6). Such a binding protein can be generally referred to herein as“a binding protein of the invention”, or preferably “an antibody orantibody fragment of the invention”.

In one embodiment, the binding protein, preferably an antibody orantibody fragment, comprises the light chain complementarity determiningregions 1, 2 and 3, comprising the amino acid sequences RASQDISNYLA (SEQID NO:1), AASSLHS (SEQ ID NO:2) and LQYSTYPIT (SEQ ID NO:3),respectively; and heavy chain complementarity determining regions 1, 2and 3, comprising the amino acid sequences NYAMS (SEQ ID NO:4),AITPSGGSTNYADSVKG (SEQ ID NO:5) and VPYRSTWYPLY (SEQ ID NO:6),respectively. The invention also provides a binding protein, preferablyan antibody or antibody fragment, that comprises the light chainvariable region shown in FIG. 1 (SEQ ID NO:7) and/or the heavy chainvariable region shown in FIG. 2 (SEQ ID NO:9).

A person skilled in the art will appreciate that the invention includesvariants to the specific binding proteins disclosed above, includingchemical equivalents to the sequences disclosed above that performsubstantially the same function as the binding proteins disclosed abovein substantially the same way. A functional variant of a binding proteinwill be able to bind to the same antigen as the binding proteinsdisclosed above. In one embodiment, the binding protein binds to glucosetransporter 8 or variants thereof, a protein comprising any one of theamino acid sequences of SEQ ID NOS:11-20, preferably SEQ ID NOS: 11-13,or a cancer-associated variant of GLUT8 that is expressed on the surfaceof cancer cells.

The inventors have discovered a novel variant of GLUT8 that is expressedon cancer cells. Accordingly, the invention includes a binding proteinthat is specific for a cancer-associated variant of glucose transporter8. In one embodiment, the cancer-associated variant of glucosetransporter 8 comprises any one of the amino acid sequences defined bySEQ ID NOS: 11-13, or a variant thereof. In another embodiment of theinvention, the cancer-associated variant of GLUT8, comprises GLUT8 thathas a modification in the N-terminal di-leucine motif. In a furtherembodiment of the invention, the N-terminal di-leucine motif has beenmodified to di-alanine.

In certain embodiments, the antibody or antibody fragment comprises allor a portion of a heavy chain constant region, such as an IgG1, IgG2,IgG3, IgG4, IgA1, IgA2, IgE, IgM or IgD constant region. Preferably, theheavy chain constant region is an IgG1 heavy chain constant region.Furthermore, the antibody or antibody fragment can comprise all or aportion of a kappa light chain constant region or a lambda light chainconstant region. Preferably, the light chain constant region is a kappalight chain constant region.

To produce human monoclonal antibodies, antibody producing cells(lymphocytes) can be harvested from a human having cancer and fused withmyeloma cells by standard somatic cell fusion procedures thusimmortalizing these cells and yielding hybridoma cells. Such techniquesare well known in the art, (e.g. the hybridoma technique originallydeveloped by Kohler and Milstein (Nature 256:495-497 (1975)) as well asother techniques such as the human B-cell hybridoma technique (Kozbor etal., Immunol. Today 4:72 (1983)), the EBV-hybridoma technique to producehuman monoclonal antibodies (Cole et al., Methods Enzymol, 121:140-67(1986)), and screening of combinatorial antibody libraries (Huse et al.,Science 246:1275 (1989)). Hybridoma cells can be screenedimmunochemically for production of antibodies specifically reactive withcancer cells and the monoclonal antibodies can be isolated.

Specific antibodies, or antibody fragments, reactive against particularantigens or molecules, such as antigens or molecules on a cancer cell,may also be generated by screening expression libraries encodingimmunoglobulin genes, or portions thereof, expressed in bacteria withcell surface components. For example, complete Fab fragments, VH regionsand FV regions can be expressed in bacteria using phage expressionlibraries (See for example Ward et al., Nature 341:544-546 (1989); Huseet al., Science 246:1275-1281 (1989); and McCafferty et al., Nature348:552-554 (1990)).

The present invention includes all antibodies and antibody fragmentsthat bind to the same antigen as the antibodies or antibody fragments ofthe invention. A person skilled in the art will appreciate that bindingassays can be used to find other antibodies and antibody fragments withthe same binding specificities as the antibodies and antibody fragmentsof the invention. As exemplified, below, a competition binding assay canbe used to find such other antibodies.

Before a competition assay is performed using flow cytometry, theminimal concentration of antibody of the invention (Ab1) that givesmaximal binding against a fixed number of cancer cells (for example,A-375 cells for VB1-050) is determined. A total of 10⁶ cells areharvested from exponentially growing cultures and incubated with variousantibody concentrations for 1 hr at 4° C. The cells are washed andincubated with a suitable detection antibody for an additional hour at4° C. After washing, the cells are analyzed by flow cytometry. For eachtest antibody, a saturation curve is generated from the data by plottingmedian fluorescence against the antibody concentration.

For the competition assay, cancer cells are prepared as above andtreated in duplicate with a fixed concentration of antibody (Ab1). Thefixed concentration is the minimal concentration of antibody thatgenerates maximal binding against a fixed number of cancer cells asdetermined above. Immediately following the addition of the Ab1, varyingconcentrations of the potential inhibitory antibody (Ab2) is added toeach tube and the mixture incubated for 1 hr at 4° C. Both the percentinhibition and change over maximum median fluorescence are calculated bysubtracting the background fluorescence (PBS-5% FCS) from the medianfluorescence reading for each test sample (Ab1+Ab2). The result is thendivided by the median fluorescence of Ab1 alone (maximal binding) minusthe background (see below). The percent of inhibition result is obtainedby multiplying by 100. The mean of the replicates along with theirrespective standard error is plotted against antibody concentration. Thefollowing formula is used to calculate the percent inhibition:PI=[(MF(_(Ab1+Ab2))−MF_(Bgd))/(MF_(Ab1)−MF_(Bgd))]×100

where PI=percent inhibition; MF(_(Ab1+Ab2))=median fluorescence measuredfor Ab1+Ab2 mixture; and MF_(Bgd)=background median fluorescence withPBS-5% FCS.

Accordingly, the invention provides a binding protein capable of bindingan antigen on a cancer cell wherein the binding protein can beidentified by a method comprising:

-   -   (1) incubating a fixed number of cancer cells with a minimal        concentration of a binding protein of the invention, preferably        an antibody or antibody fragment (Ab1) that generates maximal        binding against the fixed number of cancer cells and measuring        median fluorescence of Ab1 (MF_(Ab1));    -   (2) testing two or more concentrations of a test binding protein        (Ab2) by adding Ab2 to the Ab1 and cancer cells, and measuring        median fluorescence (MF_((Ab1+Ab2)));    -   (3) measuring background median fluorescence (MF_(bgd));    -   (4) calculating PI, wherein        PI=[(MF(_(Ab1+Ab2))−MF_(Bgd))/(MF_(Ab1)−MF_(Bgd))]×100; and    -   (5) comparing the PI to a control PI value;

wherein, a PI that has a statistically significant difference from thecontrol PI indicates that the test binding protein is capable of bindingthe antigen on the cancer cell.

A person skilled in the art will appreciate that affinity maturationtechniques could be used modify the binding proteins or immunoconjugatesof the invention by increasing its affinity for its antigen, glucosetransporter 8 or variants thereof.

Two strategies are routinely used to enhance the binding affinity of anantibody. One approach utilizes the resolution of the crystal structureof the Ab-Ag complex to identify the key residues involved in theantigen binding (Davies D. R., Cohen G. H. 1996. Interactions of proteinantigens with antibodies. Proc Natl. Acad. Sci. U.S.A. 93, 7-12).Subsequently, those residues can be mutated to enhance the interaction.The other approach mimics an in vivo antigen stimulation that drives theaffinity maturation of immunoglobulin produced by B cells. During thematuration of the immune response, the variable regions of theimmunoglobulins are subjected to somatic mutations (Mc Heyzer-WilliamsM. 2003. B-cell signaling mechanism and activation. FundamentalImmunology, Fifth edition, 195-225). This process, highly specific forthe immune system, is characterized by the introduction of pointmutations at a very high rate. It occurs only within the DNA fragmentsencoding the variable regions and excludes the conserved domains. The Bcells expressing the somatically mutated variant antibody are thensubjected to an antigen-mediated selection resulting in the selection ofhigher affinity immunoglobulin. In order to replicate this phenomenon invitro, several approaches have been used to introduce mutations eitherby random or targeted processes. The random mutations can be introducedusing error-prone PCR, chain shuffling or mutator E. coli strains(Clackson T. Hoogenboom N. R., Griffiths A. D. and Winter G. 1991 Makingantibody fragments using phage display libraries. Nature 352, 624-628,Hawkins R. E., Russell S. J. and Winter G. 1992. Selection of phageantibodies by binding affinity. Mimicking affinity maturation. J. Mol.Biol. 226, 889-896, Low N., Holliger P. and Winter G. 1996. Mimickingsomatic hypermutation: affinity maturation of antibodies displayed onbacteriophage using a bacterial mutator strain. J. Mol. Biol. 260,359-368). This strategy leads to the creation of large libraries inwhich reactive clones are selected with a display technology such asribosome, phage or yeast (Min L. (2000). Applications of displaytechnology in protein analysis. Nat. Biotechnol. 18, 1251-1256).

The targeted mutations of the CDRs, especially CDR3 of the light andheavy chains, have been shown to be an effective technique forincreasing antibody affinity. Blocks of 3 to 4 amino acids of the CDR3or specific regions called “hot-spots” are targeted for mutagenesis.Yang et al reported an increase of 420 fold of an anti-HIV gp120 Fabfragment by mutating four CDR residues (Yang W. P., Green K.,Pinz-Sweeney S., Briones A. T., Burton D. R. and Barbas C. F. III. 1995.CDR walking mutagenesis for the affinity maturation of a potent humananti-HIV-1 antibody into picomolar range. J. Mol. Biol., 254, 392-403).One mutation in the VL CDR3 combined with three mutations in the VH CDR3of the C6.5 scFv yielded a 1230 fold increased affinity (Schier R.,McCall A., Adams G. P., Marshall K. W., Merrit H., Yin M., Crawford R.S. Weiner L. M., Marks C. and Marks J. D. 1996. Isolation of picomolaraffinity anti-c-erbB-2 single-chain Fv by molecular evolution of thecomplementary determining regions in the center of the antibody bindingsite. J. Mol. Biol., 263, 551-567).

“Hot spots” are the sequences where somatic hypermutation takes place invivo (Neuberger M. S. and Milstein C. 1995. Somatic hypermutation. Curr.Opin. Immunol. 7, 248-254). The hotspot sequences can be defined asconsensus nucleotide sequences in certain codons. The consensus sequenceis the tetranucleotide, RGYW (SEQ ID NO:31), in which R can be either Aor G, Y can be C or T and W can be either A or T (Neuberger M. S andMilstein C. 1995. Somatic hypermutation. Curr. Opin. Immunol. 7,248-254). In addition, the serine residues encoded by the nucleotidesAGY are predominantly present in the CDRs regions of the variable domainover those encoded by TCN corresponding to a potential hot-spotsequences (Wagner S. D., Milstein C. and Neuberger M. S. 1995. Codonbias targets mutation. Nature, 376, 732). The structural analysis hasshown that the CDR loops contribute the most to the antigen binding,especially the CDR3 loops (Giudicelli V., Chaume D. and Lefranc M. P.2004. IMGT/V-QUEST, an integrated software program for immunoglobulinand T cell receptor V-J and V-D-J rearrangement analysis. Nucleic AcidsRes. 32, 435-440). Therefore, the nucleotide sequence of the CDRs of theheavy and light chains of each antibody of the invention is scanned forthe presence of the hot-spot sequences and AGY codons. The identifiedhot-spots of the CDR regions of the light and heavy chain are comparedto the germinal sequences of the heavy and light chains using theInternational ImMunoGen Tics database (IMGT,imgt.cines.fr/textes/vquest/) (Davies D. R., Padlan E. A. and Sheriff S.1990. Antibody-antigen complexes. Annu. Rev. Biochem. 59, 439-473). Asequence, identical to the germ line, suggest that somatic mutation hasnot occurred; therefore the random mutations are introduced mimickingthe somatic events occurring in vivo. In contrast, a different sequenceshows that some somatic mutations have already occurred. It will remainto be determined if the in vivo somatic mutation was optimal. Thehot-spots that code for buried or conserved amino acids within the CDRsare not mutagenized. These residues are usually critical for the overallstructure and are unlikely to interact with the antigen since they areburied. In addition, the sequences can be compared to the predictedlocations in the germ line sequences where somatic mutations occurredpredominantly (Tomlinson I. M., Cox J. P. L., Gherardi E., Lesk A. M.and Chotia C. 1995. The structural repertoire of the human Vldomain.EMBO J. 14, 4628-4638, Tomlinson I. M., Walter G., Jones P. T., Dear P.N., Sonnhammer E. L. L. and Winter G. 1996. The imprint of somatichypermutation on the repertoire of human germline V genes. J. Mol. Biol.256, 813-817). A similar strategy was applied for the affinitymaturation of BL22 scFv. A point mutation introduced in the CDR3 of theheavy resulted in 5 to 10 fold increase in binding activity on variousCD22-positive cell lines (Salvatore G., Beers R., Margulies I., KreitmanR. J. and Pastan I. 2002. Improved cytotoxic activity toward cell linesand fresh leukemia cells of a mutant anti-CD22 immunotoxin obtained byantibody phage display. Clinical Cancer research, 8, 995-1002). Also,the mutation of various amino acids in the CDR1 and CDR2 loops alsoproduced mutant with increase affinity ranging from 3 fold to 7 fold (HoM., Kreitman J., Onda M. and Pastan I. 2005. In vitro antibody evolutiontargeting germline hot spots to increase activity of an anti-CD22immunotoxin. J. Biol. Chem., 280, 607-617).

After mutations are introduced, either by random or targeted processes,the antibodies are expressed and assessed for function. For instance,functional screening can be based on binding. Once function is assessed,then DNA sequencing of the chosen antibodies can be carried out usingknown methods.

In another embodiment, the anchored periplasmic expression (APEx) methoddescribed by Harvey, B et al (PNAS 2004 Jun. 22; 101(25): 9193-8) isused for affinity maturation of the binding proteins or immunoconjugatesof the invention.

Accordingly, the invention includes binding proteins of the inventionthat have been affinity maturized to increase the affinity of thebinding protein to glucose transporter 8 or variants thereof; a proteincomprising amino acid sequences of SEQ ID NOS:11-20, preferably SEQ IDNOS:11-13; or a cancer-associated variant of glucose transporter 8.

The invention also provides compositions comprising the binding proteinsof the invention, preferably antibodies and antibody fragments, with apharmaceutically acceptable excipient, carrier, buffer or stabilizer.

(iii) Novel Cancer-Associated Antigens

As mentioned above, the inventors have identified the antigen to whichthe binding proteins of the invention bind. The novel-cancer associatedantigen is expressed on the surface of cancer cells and is notsignificantly expressed on the surface of normal cells. Accordingly, theinvention includes an isolated protein that can specifically bind withone of the binding proteins of the invention, and nucleic acid sequencesand uses thereof.

In one embodiment, the invention provides an isolated protein comprisingglucose transporter 8 or variants thereof. In another embodiment, theinvention provides an isolated protein comprising any one of the aminoacid sequences of SEQ ID NOS: 11-20 or variants thereof. In a furtherembodiment, the invention provides an isolated protein comprising theamino acid sequence of SEQ ID NO:11 or a variant thereof. The inventionalso provides an isolated protein comprising the amino acid sequence ofSEQ ID NO:12 or a variant thereof. Further, the invention provides anisolated protein comprising the amino acid sequence of SEQ ID NO:13 or avariant thereof. In addition, the invention provides a cancer-associatedvariant of glucose transporter 8 that is expressed on the surface ofcancer cells. In one embodiment of the invention, the cancer-associatedvariant of GLUT8, comprises the amino acid sequence defined by any oneof SEQ ID NOS: 11, 12 or 13, or variants thereof. In another embodimentof the invention, the cancer-associated variant of GLUT8, comprisesGLUT8 that has a modification in the N-terminal di-leucine motif. In afurther embodiment of the invention, the N-terminal di-leucine motif hasbeen modified to di-alanine.

A person skilled in the art will appreciate that the invention includesvariants to the amino acid sequences of SEQ ID NOS:11-13, includingchemical equivalents to the sequences disclosed by the presentinvention. Such equivalents include proteins that perform substantiallythe same function as the specific proteins disclosed herein insubstantially the same way. For example, equivalents include, withoutlimitation, conservative amino acid substitutions.

In one embodiment, the variant amino acid sequences of the isolatedproteins of the invention have at least 50%, preferably at least 60%,more preferably at least 70%, most preferably at least 80%, and evenmore preferably at least 90% sequence identity to SEQ ID NOS:11-13.

The invention includes the use of the isolated protein. For example, theuse of the isolated proteins of the invention to generate bindingproteins and immunoconjugates that can be used to treat or preventcancer or that can be used to detect or monitor cancer in a subject.Accordingly, the invention includes the use of the isolated proteins ofthe invention in the manufacture of a medicament to treat or preventcancer.

(C) Immunoconjugates

The invention also includes an immunoconjugate comprising (1) a bindingprotein of the invention, preferably an antibody or antibody fragment,that has been attached to (2) an effector molecule. In one embodiment,the binding protein of the invention binds to an antigen or molecule ona cancer cell.

The antigen can be glucose transporter 8 or variants thereof; a proteincomprising any one of the amino acid sequences defined by SEQ ID NOS:11-20, preferably SEQ ID NOS:11, 12 or 13; or a cancer-associatedvariant of glucose transporter 8. In one embodiment of the invention,the cancer-associated variant of GLUT8, comprises the amino acidsequence defined by any one of SEQ ID NOS: 11, 12 or 13, or variantsthereof. In another embodiment of the invention, the cancer-associatedvariant of GLUT8, comprises GLUT8 that has a modification in theN-terminal di-leucine motif. In a further embodiment of the invention,the N-terminal di-leucine motif has been modified to di-alanine.

In a preferred, embodiment the effector molecule is (i) a label, whichcan generate a detectable signal, directly or indirect, or (ii) a cancertherapeutic agent, which is either cytotoxic, cytostatic or otherwiseprevents or reduces the ability of the cancer cells to divide and/ormetastasize. Such an immunoconjugate can be generally referred to as“the immunoconjugate of the invention” herein.

In an embodiment of the invention, the effector molecule is a cancertherapeutic agent. The cancer therapeutic agent is preferably a toxinthat is either cytotoxic, cytostatic or otherwise prevents or reducesthe ability of the cancer cells to divide and/or metastasize.Accordingly, one aspect of the invention is an immunoconjugatecomprising (1) a binding protein of the invention, preferably anantibody or antibody fragment, attached to (2) a cancer therapeuticagent, such as a cytotoxin.

In another embodiment, the immunoconjugate is internalized and thecancer therapeutic agent is a cytotoxin that blocks the proteinsynthesis of the cell, therein leading to cell death. Importantly, sincemost normal cells do not widely express the antigen present on thecancer cells, they cannot bind and internalize the immunoconjugate, andare protected from the killing effect of the toxin or other cancertherapeutic agents.

A variety of effector molecules may be used in the immunoconjugates ofthe invention and a number of such effector molecules areintracellularly active molecules. Accordingly, in an embodiment of theinvention, the immunoconjugate is internalized by the cancer cell.

In preferred embodiments, the effector molecule is a cancer therapeuticagent, more preferably a cytotoxin that comprises a polypeptide havingribosome-inactivating activity including, without limitation, gelonin,bouganin, saporin, ricin, ricin A chain, bryodin, diphtheria toxin,restrictocin, Pseudomonas exotoxin A and variants thereof. When theprotein is a ribosome-inactivating protein, the immunoconjugate must beinternalized upon binding to the cancer cell in order for the protein tobe cytotoxic to the cells. Accordingly, in an embodiment of theinvention, the effector molecule is a cytotoxin and the immunoconjugateis internalized by the cancer cell.

In one embodiment of the invention, the toxin is bouganin or Pseudomonasexotoxin A, and variants thereof. In another embodiment, the toxin ismodified bouganin or a truncated form of Pseudomonas exotoxin A thatlacks the cell binding domain. In a further embodiment, the toxin is abouganin substantially devoid of T-cell epitopes or a truncated form ofPseudomonas exotoxin A that consists of amino acids 252-608.

In other nonlimiting embodiments, the cancer therapeutic agent comprisesan agent that acts to disrupt DNA. Thus, the cancer therapeutic agentsmay be selected, without limitation, from enediynes (e.g., calicheamicinand esperamicin) and non-enediyne small molecule agents (e.g.,bleomycin, methidiumpropyl-EDTA-Fe(II)). Other cancer therapeutic agentsuseful in accordance with the invention include, without limitation,daunorubicin, doxorubicin, distamycin A, cisplatin, mitomycin C,ecteinascidins, duocarmycin/CC-1065, and bleomycin/pepleomycin.

In other nonlimiting embodiments, the cancer therapeutic agent comprisesan agent that acts to disrupt tubulin. Such agents may comprise, withoutlimitation, rhizoxin/maytansine, paclitaxel, vincristine andvinblastine, colchicine, auristatin dolastatin 10 MMAE, and pelorusideA.

In other nonlimiting embodiments, the cancer therapeutic portion of animmunoconjugate of the invention may comprise an alkylating agentincluding, without limitation, Asaley NSC 167780, AZQ NSC 182986, BCNUNSC 409962, Busulfan NSC 750, carboxyphthalatoplatinum NSC 271674, CBDCANSC 241240, CCNU NSC 79037, CHIP NSC 256927, chlorambucil NSC 3088,chlorozotocin NSC 178248, cis-platinum NSC 119875, clomesone NSC 338947,cyanomorpholinodoxorubicin NSC 357704, cyclodisone NSC 348948,dianhydrogalactitol NSC 132313, fluorodopan NSC 73754, hepsulfam NSC329680, hycanthone NSC 142982, melphalan NSC 8806, methyl CCNU NSC95441, mitomycin C NSC 26980, mitozolamide NSC 353451, nitrogen mustardNSC 762, PCNU NSC 95466, piperazine NSC 344007, piperazinedione NSC135758, pipobroman NSC 25154, porfiromycin NSC 56410, spirohydantoinmustard NSC 172112, teroxirone NSC 296934, tetraplatin NSC 363812,thio-tepa NSC 6396, triethylenemelamine NSC 9706, uracil nitrogenmustard NSC 34462, and Yoshi-864 NSC 102627.

In other nonlimiting embodiments, the cancer therapeutic agent portionof the immunoconjugate of the invention may comprise an antimitoticagent including, without limitation, allocoichicine NSC 406042,Halichondrin B NSC 609395, colchicine NSC 757, colchicine derivative NSC33410, dolastatin 10 NSC 376128 (NG—auristatin derived), maytansine NSC153858, rhizoxin NSC 332598, taxol NSC 125973, taxol derivative NSC608832, thiocolchicine NSC 361792, trityl cysteine NSC 83265,vinblastine sulfate NSC 49842, and vincristine sulfate NSC 67574.

In other nonlimiting embodiments, the cancer therapeutic agent portionof the immunoconjugate of the invention may comprise an topoisomerase Iinhibitor including, without limitation, camptothecin NSC 94600,camptothecin, Na salt NSC 100880, aminocamptothecin NSC 603071,camptothecin derivative NSC 95382, camptothecin derivative NSC 107124,camptothecin derivative NSC 643833, camptothecin derivative NSC 629971,camptothecin derivative NSC 295500, camptothecin derivative NSC 249910,camptothecin derivative NSC 606985, camptothecin derivative NSC 374028,camptothecin derivative NSC 176323, camptothecin derivative NSC 295501,camptothecin derivative NSC 606172, camptothecin derivative NSC 606173,camptothecin derivative NSC 610458, camptothecin derivative NSC 618939,camptothecin derivative NSC 610457, camptothecin derivative NSC 610459,camptothecin derivative NSC 606499, camptothecin derivative NSC 610456,camptothecin derivative NSC 364830, camptothecin derivative NSC 606497,and morpholinodoxorubicin NSC 354646.

In other nonlimiting embodiments, cancer therapeutic agent portion ofthe immunoconjugate of the invention may comprise an topoisomerase IIinhibitor including, without limitation, doxorubicin NSC 123127,amonafide NSC 308847, m-AMSA NSC 249992, anthrapyrazole derivative NSC355644, pyrazoloacridine NSC 366140, bisantrene HCL NSC 337766,daunorubicin NSC 82151, deoxydoxorubicin NSC 267469, mitoxantrone NSC301739, menogaril NSC 269148, N,N-dibenzyl daunomycin NSC 268242,oxanthrazole NSC 349174, rubidazone NSC 164011, VM-26 NSC 122819, andVP-16 NSC 141540.

In other nonlimiting embodiments, the cancer therapeutic agent portionof the immunoconjugate of the invention may comprise an RNA or DNAantimetabolite including, without limitation, L-alanosine NSC 153353,5-azacytidine NSC 102816, 5-fluorouracil NSC 19893, acivicin NSC 163501,aminopterin derivative NSC 132483, aminopterin derivative NSC 184692,aminopterin derivative NSC 134033, an antifol NSC 633713, an antifol NSC623017, Baker's soluble antifol NSC 139105, dichlorallyl lawsone NSC126771, brequinar NSC 368390, ftorafur (pro-drug) NSC 148958,5,6-dihydro-5-azacytidine NSC 264880, methotrexate NSC 740, methotrexatederivative NSC 174121, N-(phosphonoacetyl)-L-aspartate (PALA) NSC224131, pyrazofurin NSC 143095, trimetrexate NSC 352122, 3-HP NSC 95678,2′-deoxy-5-fluorouridine NSC 27640, 5-HP NSC 107392, alpha-TGDR NSC71851, aphidicolin glycinate NSC 303812, ara-C NSC 63878,5-aza-2′-deoxycytidine NSC 127716, beta-TGDR NSC 71261, cyclocytidineNSC 145668, guanazole NSC 1895, hydroxyurea NSC 32065, inosineglycodialdehyde NSC 118994, macbecin II NSC 330500, pyrazoloimidazoleNSC 51143, thioguanine NSC 752, and thiopurine NSC 755.

In another nonlimiting embodiment, the therapeutic portion of theimmunoconjugates may be a nucleic acid. Nucleic acids that may be usedinclude, but are not limited to, anti-sense RNA, genes or otherpolynucleotides, nucleic acid analogs such as thioguanine andthiopurine.

The present invention further provides immunoconjugates comprising (i) abinding protein of the invention, preferably an antibody or antibodyfragment, attached to (2) an effector molecule, wherein the effectormolecule is a label, which can generate a detectable signal, indirectlyor directly. These immunoconjugates can be used for research ordiagnostic applications, such as for the in vivo detection of cancer.The label is preferably capable of producing, either directly orindirectly, a detectable signal. For example, the label may beradio-opaque or a radioisotope, such as ³H, ¹⁴C, ³²P, ³⁵S, ¹²³I, ¹²⁵I,¹³¹I; a fluorescent (fluorophore) or chemiluminescent (chromophore)compound, such as fluorescein isothiocyanate, rhodamine or luciferin; anenzyme, such as alkaline phosphatase, beta-galactosidase or horseradishperoxidase; an imaging agent; or a metal ion.

In another embodiment, the immunoconjugate is detectable indirectly. Forexample, a secondary antibody that is specific for the immunoconjugateand contains a detectable label can be used to detect theimmunoconjugate.

The binding protein of the invention, preferably an antibody or antibodyfragment, may be “attached to” the effector molecule by any means bywhich the binding protein can be associated with, or linked to, theeffector molecule. For example, the binding protein may be attached tothe effector molecule by chemical or recombinant means. Chemical meansfor preparing fusions or conjugates are known in the art and can be usedto prepare the immunoconjugate. The method used to conjugate the bindingprotein and effector molecule must be capable of joining the bindingprotein with the effector molecule without interfering with the abilityof the binding protein to bind to the antigen on the cancer cell.

The binding protein of the invention may be linked indirectly to theeffector molecule. For example, the binding protein may be directlylinked to a liposome containing the effector molecule of one of severaltypes. The effector molecule(s) and/or binding protein may also be boundto a solid surface.

In one embodiment, the binding protein, preferably an antibody orantibody fragment, and effector molecule are both proteins and can beconjugated using techniques well known in the art. There are severalhundred crosslinkers available that can conjugate two proteins. (See forexample “Chemistry of Protein Conjugation and Crosslinking”. 1991, ShansWong, CRC Press, Ann Arbor). The crosslinker is generally chosen basedon the reactive functional groups available or inserted on the bindingprotein, preferably an antibody or antibody fragment, and/or effectormolecule. In addition, if there are no reactive groups, aphotoactivatible crosslinker can be used. In certain instances, it maybe desirable to include a spacer between the binding protein, preferablyan antibody or antibody fragment, and effector molecule. Crosslinkingagents known to the art include the homobifunctional agents:glutaraldehyde, dimethyladipimidate and Bis(diazobenzidine) and theheterobifunctional agents: m Maleimidobenzoyl-N-Hydroxysuccinimide andSulfo-m Maleimidobenzoyl-N-Hydroxysuccinimide.

A binding protein-effector molecule protein fusion may also be preparedusing recombinant DNA techniques. In such a case a DNA sequence encodingthe binding protein is fused to a DNA sequence encoding the effectormolecule, resulting in a chimeric DNA molecule. The chimeric DNAsequence is transfected into a host cell that expresses the fusionprotein. The fusion protein can be recovered from the cell culture andpurified using techniques known in the art.

Examples of attaching an effector molecule, which is a label, to thebinding protein include the methods described in Hunter, et al., Nature144:945 (1962); David, et al., Biochemistry 13:1014 (1974); Pain, etal., J. Immunol. Meth. 40:219 (1981); Nygren, J. Histochem. andCytochem. 30:407 (1982); Wensel and Meares, Radioimmunoimaging AndRadioimmunotherapy, Elsevier, N.Y. (1983); and Colcher et al., “Use OfMonoclonal Antibodies As Radiopharmaceuticals For The Localization OfHuman Carcinoma Xenografts In Athymic Mice”, Meth. Enzymol., 121:802-16(1986).

(D) Preparation of Proteins of the Invention

A person skilled in the art will appreciate that the proteins of theinvention, such as the light and heavy complementarity determiningregions, the light and heavy chain variable regions, antibodies andantibody fragments, immunoconjugates and novel cancer-associatedantigens of the invention, may be prepared in any of several ways, butis most preferably prepared using recombinant methods.

Accordingly, the nucleic acid molecules of the present invention may beincorporated in a known manner into an appropriate expression vectorwhich ensures good expression of the proteins of the invention. Possibleexpression vectors include but are not limited to cosmids, plasmids, ormodified viruses (e.g. replication defective retroviruses, adenovirusesand adeno-associated viruses), so long as the vector is compatible withthe host cell used. The expression vectors are “suitable fortransformation of a host cell”, which means that the expression vectorscontain a nucleic acid molecule of the invention and regulatorysequences selected on the basis of the host cells to be used forexpression, which is operatively linked to the nucleic acid molecule.Operatively linked is intended to mean that the nucleic acid is linkedto regulatory sequences in a manner which allows expression of thenucleic acid.

The invention therefore contemplates a recombinant expression vector ofthe invention containing a nucleic acid molecule of the invention, or afragment thereof, and the necessary regulatory sequences for thetranscription and translation of the inserted protein-sequence.

Suitable regulatory sequences may be derived from a variety of sources,including bacterial, fungal, viral, mammalian, or insect genes (Forexample, see the regulatory sequences described in Goeddel, GeneExpression Technology Methods in Enzymology 185, Academic Press, SanDiego, Calif. (1990)). Selection of appropriate regulatory sequences isdependent on the host cell chosen as discussed below, and may be readilyaccomplished by one of ordinary skill in the art. Examples of suchregulatory sequences include: a transcriptional promoter and enhancer orRNA polymerase binding sequence, a ribosomal binding sequence, includinga translation initiation signal. Additionally, depending on the hostcell chosen and the vector employed, other sequences, such as an originof replication, additional DNA restriction sites, enhancers, andsequences conferring inducibility of transcription may be incorporatedinto the expression vector.

The recombinant expression vectors of the invention may also contain aselectable marker gene which facilitates the selection of host cellstransformed or transfected with a recombinant molecule of the invention.Examples of selectable marker genes are genes encoding a protein such asG418 and hygromycin which confer resistance to certain drugs,β-galactosidase, chloramphenicol acetyltransferase, firefly luciferase,or an immunoglobulin or portion thereof such as the Fc portion of animmunoglobulin preferably IgG. Transcription of the selectable markergene is monitored by changes in the concentration of the selectablemarker protein such as β-galactosidase, chloramphenicolacetyltransferase, or firefly luciferase. If the selectable marker geneencodes a protein conferring antibiotic resistance such as neomycinresistance transformant cells can be selected with G418. Cells that haveincorporated the selectable marker gene will survive, while the othercells die. This makes it possible to visualize and assay for expressionof recombinant expression vectors of the invention and in particular todetermine the effect of a mutation on expression and phenotype. It willbe appreciated that selectable markers can be introduced on a separatevector from the nucleic acid of interest.

The recombinant expression vectors may also contain genes which encode afusion moiety which provides increased expression of the recombinantprotein; increased solubility of the recombinant protein; and aid in thepurification of the target recombinant protein by acting as a ligand inaffinity purification. For example, a proteolytic cleavage site may beadded to the target recombinant protein to allow separation of therecombinant protein from the fusion moiety subsequent to purification ofthe fusion protein. Typical fusion expression vectors include pGEX(Amrad Corp., Melbourne, Australia), pMal (New England Biolabs, Beverly,Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) which fuse glutathioneS-transferase (GST), maltose E binding protein, or protein A,respectively, to the recombinant protein.

Recombinant expression vectors can be introduced into host cells toproduce a transformed host cell. The terms “transformed with”,“transfected with”, “transformation” and “transfection” are intended toencompass introduction of nucleic acid (e.g. a vector) into a cell byone of many possible techniques known in the art. The term “transformedhost cell” as used herein is intended to also include cells capable ofglycosylation that have been transformed with a recombinant expressionvector of the invention. Prokaryotic cells can be transformed withnucleic acid by, for example, electroporation or calcium-chloridemediated transformation. For example, nucleic acid can be introducedinto mammalian cells via conventional techniques such as calciumphosphate or calcium chloride co-precipitation, DEAE-dextran mediatedtransfection, lipofectin, electroporation or microinjection. Suitablemethods for transforming and transfecting host cells can be found inSambrook et al. (Molecular Cloning: A Laboratory Manual, 3rd Edition,Cold Spring Harbor Laboratory Press, 2001), and other laboratorytextbooks.

Suitable host cells include a wide variety of eukaryotic host cells andprokaryotic cells. For example, the proteins of the invention may beexpressed in yeast cells or mammalian cells. Other suitable host cellscan be found in Goeddel, Gene Expression Technology: Methods inEnzymology 185, Academic Press, San Diego, Calif. (1991). In addition,the proteins of the invention may be expressed in prokaryotic cells,such as Escherichia coli (Zhang et al., Science 303(5656): 371-3(2004)). In addition, a Pseudomonas based expression system such asPseudomonas fluorescens can be used (US Patent Application PublicationNo. US 2005/0186666, Schneider, Jane C et al.).

Yeast and fungi host cells suitable for carrying out the presentinvention include, but are not limited to Saccharomyces cerevisiae, thegenera Pichia or Kluyveromyces and various species of the genusAspergillus. Examples of vectors for expression in yeast S. cerevisiaeinclude pYepSec1 (Baldari. et al., Embo J. 6:229-234 (1987)), pMFa.(Kurjan and Herskowitz, Cell 30:933-943 (1982)), pJRY 88 (Schultz etal., Gene 54:113-123 (1987)), and pYES2 (Invitrogen Corporation, SanDiego, Calif.). Protocols for the transformation of yeast and fungi arewell known to those of ordinary skill in the art (see Hinnen et al.,Proc. Natl. Acad. Sci. USA 75:1929 (1978); Itoh et al., J. Bacteriology153:163 (1983), and Cullen et al. (Bio/Technology 5:369 (1987)).

Mammalian cells suitable for carrying out the present invention include,among others: COS (e.g., ATCC No. CRL 1650 or 1651), BHK (e.g. ATCC No.CRL 6281), CHO (ATCC No. CCL 61), HeLa (e.g., ATCC No. CCL 2), 293 (ATCCNo. 1573) and NS-1 cells. Suitable expression vectors for directingexpression in mammalian cells generally include a promoter (e.g.,derived from viral material such as polyoma, Adenovirus 2,cytomegalovirus and Simian Virus 40), as well as other transcriptionaland translational control sequences. Examples of mammalian expressionvectors include pCDM8 (Seed, B., Nature 329:840 (1987)) and pMT2PC(Kaufman et al., EMBO J. 6:187-195 (1987)).

Given the teachings provided herein, promoters, terminators, and methodsfor introducing expression vectors of an appropriate type into plant,avian, and insect cells may also be readily accomplished. For example,within one embodiment, the proteins of the invention may be expressedfrom plant cells (see Sinkar et al., J. Biosci (Bangalore) 11:47-58(1987), which reviews the use of Agrobacterium rhizogenes vectors; seealso Zambryski et al., Genetic Engineering, Principles and Methods,Hollaender and Setlow (eds.), Vol. VI, pp. 253-278, Plenum Press, NewYork (1984), which describes the use of expression vectors for plantcells, including, among others, PAPS2022, PAPS2023, and PAPS2034).

Insect cells suitable for carrying out the present invention includecells and cell lines from Bombyx, Trichoplusia or Spodotera species.Baculovirus vectors available for expression of proteins in culturedinsect cells (SF 9 cells) include the pAc series (Smith et al., Mol.Cell. Biol. 3:2156-2165 (1983)) and the pVL series (Lucklow, V. A., andSummers, M. D., Virology 170:31-39 (1989)). Some baculovirus-insect cellexpression systems suitable for expression of the recombinant proteinsof the invention are described in PCT/US/02442.

Alternatively, the proteins of the invention may also be expressed innon-human transgenic animals such as rats, rabbits, sheep and pigs(Hammer et al. Nature 315:680-683 (1985); Palmiter et al. Science222:809-814 (1983); Brinster et al. Proc. Natl. Acad. Sci. USA82:4438-4442 (1985); Palmiter and Brinster Cell 41:343-345 (1985) andU.S. Pat. No. 4,736,866).

The proteins of the invention may also be prepared by chemical synthesisusing techniques well known in the chemistry of proteins such as solidphase synthesis (Merrifield, J. Am. Chem. Assoc. 85:2149-2154 (1964);Frische et al., J. Pept. Sci. 2(4): 212-22 (1996)) or synthesis inhomogenous solution (Houbenweyl, Methods of Organic Chemistry, ed. E.Wansch, Vol. 15 I and II, Thieme, Stuttgart (1987)).

N-terminal or C-terminal fusion proteins comprising the proteins of theinvention conjugated with other molecules, such as proteins may beprepared by fusing, through recombinant techniques. The resultant fusionproteins contain a protein of the invention fused to the selectedprotein or marker protein as described herein. The recombinant proteinof the invention may also be conjugated to other proteins by knowntechniques. For example, the proteins may be coupled usingheterobifunctional thiol-containing linkers as described in WO 90/10457,N-succinimidyl-3-(2-pyridyldithio-proprionate) or N-succinimidyl-5thioacetate. Examples of proteins which may be used to prepare fusionproteins or conjugates include cell binding proteins such asimmunoglobulins, hormones, growth factors, lectins, insulin, low densitylipoprotein, glucagon, endorphins, transferrin, bombesin,asialoglycoprotein glutathione-S-transferase (GST), hemagglutinin (HA),and truncated myc.

Accordingly, the invention provides a recombinant expression vectorcomprising the nucleic acid sequences that encode the proteins of theinvention, such as the light and heavy chain complementarity determiningregions, the light and heavy chain variable regions, the bindingproteins, such as antibodies and antibody fragments, immunoconjugates ofthe invention and novel isolated proteins of the invention. Further, theinvention provides a host cell comprising the recombinant expressionvector of the invention.

(E) Therapeutic Methods and Pharmaceutical Compositions of the BindingProteins and Immunotoxins of the Invention

The inventors have shown that the binding proteins of the invention bindto glucose transporter 8 or variants thereof; a protein comprising anyone of the amino acid sequences defined by SEQ ID NOS:11-20, preferably11, 12 or 13; or a cancer-associated variant of glucose transporter 8.In one embodiment of the invention, the cancer-associated variant ofGLUT8, comprises the amino acid sequence defined by any one of SEQ IDNOS: 11, 12 or 13, or variants thereof. In another embodiment of theinvention, the cancer-associated variant of GLUT8, comprises GLUT8 thathas a modification in the N-terminal di-leucine motif. In a furtherembodiment of the invention, the N-terminal di-leucine motif has beenmodified to di-alanine.

In addition, the inventors have shown that the binding proteins of theinvention show specificity for cancer cells and that they areinternalized by the cell. Thus, the binding proteins of the inventioncan be used for the targeted delivery of bioactive or medically relevantagents, such as imaging, radioactive or cytotoxic agents.

In one embodiment, the invention provides a method of treating orpreventing cancer, comprising administering to a subject having orsuspected of having cancer an effective amount of the immunoconjugate ofthe invention. In another embodiment, the invention provides the use ofan effective amount of the immunoconjugate of the invention for themanufacture of a medicament for treating or preventing cancer.Furthermore, the invention provides the use of an effective amount ofthe immunoconjugate of the invention, further comprising the use of anadditional cancer therapeutic agent for the manufacture of a medicamentfor simultaneous, separate or sequential treatment or prevention ofcancer. The invention also provides the use of an effective amount ofthe immunoconjugate of the invention for treating or preventing cancer.Further, the invention provides the use of an effective amount of theimmunoconjugate of the invention, further comprising the use of anadditional cancer therapeutic agent for simultaneous, separate orsequential treatment or prevention of cancer.

In one embodiment of the invention, cancer includes, without limitation,stomach cancer, colon cancer, prostate cancer as well as cervicalcancer, uterine cancer, ovarian cancer, pancreatic cancer, kidneycancer, liver cancer, head and neck cancer, squamous cell carcinoma,gastrointestinal cancer, breast cancer (such as carcinoma, ductal,lobular, and nipple), lung cancer, non-Hodgkin's lymphoma, multiplemyeloma, leukemia (such as acute lymphocytic leukemia, chroniclymphocytic leukemia, acute myelogenous leukemia, and chronicmyelogenous leukemia), brain cancer, neuroblastoma, sarcomas, rectumcancer, bladder cancer, pancreatic cancer, endometrial cancer,plasmacytoma, lymphoma, and melanoma. In a preferred embodiment, thecancer includes, without limitation, breast cancer, prostate cancer,colon cancer, bladder cancer, cervical cancer, kidney cancer, melanoma,liver cancer, ovarian cancer, pancreatic cancer, stomach cancer, andhead and neck cancer.

The ability of the immunoconjugate of the invention to selectivelyinhibit or destroy cells having cancer may be readily tested in vitrousing cancer cell lines. The selective inhibitory effect of theimmunoconjugates of the invention may be determined, for example bydemonstrating the selective inhibition of cellular proliferation of thecancer cells.

Toxicity may also be measured based on cell viability, for example, theviability of cancer and normal cell cultures exposed to theimmunoconjugate may be compared. Cell viability may be assessed by knowntechniques, such as trypan blue exclusion assays.

In another example, a number of models may be used to test theeffectiveness of the immunoconjugates of the invention. Thompson, E. W.et al. (Breast Cancer Res. Treatment 31:357-370 (1994)) has described amodel for the determination of invasiveness of human breast cancer cellsin vitro by measuring tumor cell-mediated proteolysis of extracellularmatrix and tumor cell invasion of reconstituted basement membrane(collagen, laminin, fibronectin, Matrigel or gelatin). Other applicablecancer cell models include cultured ovarian adenocarcinoma cells (Young,T. N. et al. Gynecol. Oncol. 62:89-99 (1996); Moore, D. H. et al.Gynecol. Oncol. 65:78-82 (1997)), human follicular thyroid cancer cells(Demeure, M. J. et al., World J. Surg. 16:770-776 (1992)), humanmelanoma (A-2058) and fibrosarcoma (HT-1080) cell lines (Mackay, A. R.et al. Lab. Invest. 70:781 783 (1994)), and lung squamous (HS-24) andadenocircinoma (SB-3) cell lines (Spiess, E. et al. J. Histochem.Cytochem. 42:917-929 (1994)). An in vivo test system involving theimplantation of tumors and measurement of tumor growth and metastasis inathymic nude mice has also been described (Thompson, E. W. et al.,Breast Cancer Res. Treatment 31:357-370 (1994); Shi, Y. E. et al.,Cancer Res. 53:1409-1415 (1993)).

The immunoconjugates of the invention may be formulated intopharmaceutical compositions for administration to subjects in abiologically compatible form suitable for administration in vivo. Thesubstances may be administered to living organisms including humans, andanimals. Administration of a therapeutically active amount of thepharmaceutical compositions of the present invention is defined as anamount effective, at dosages and for periods of time necessary toachieve the desired result. For example, a therapeutically active amountof a substance may vary according to factors such as the disease state,age, sex, and weight of the individual, and the ability of therecombinant protein of the invention to elicit a desired response in theindividual. Dosage regime may be adjusted to provide the optimumtherapeutic response. For example, several divided doses may beadministered daily or the dose may be proportionally reduced asindicated by the exigencies of the therapeutic situation.

Accordingly, the present invention provides a pharmaceutical compositionfor treating or preventing cancer comprising the immunoconjugates of theinvention, and a pharmaceutically acceptable carrier, diluent orexcipient. In a preferred embodiment, the effector molecule of theimmunoconjugate in the pharmaceutical composition is a cancertherapeutic agent, more preferably a toxin.

The pharmaceutical preparation comprising the immunoconjugate of theinvention may be administered systemically. The pharmaceuticalpreparation may be administered directly to the cancer site. Dependingon the route of administration, the immunoconjugate may be coated in amaterial to protect the compound from the action of enzymes, acids andother natural conditions that may inactivate the compound.

In accordance with one aspect of the present invention, theimmunoconjugate is delivered to the patient by direct administration.The invention contemplates the pharmaceutical composition beingadministered in at least an amount sufficient to achieve the endpoint,and if necessary, comprises a pharmaceutically acceptable carrier.

The invention also provides methods for reducing the risk ofpost-surgical complications comprising administering an effective amountof the immunoconjugate of the invention before, during, or after surgeryto treat cancer.

The compositions described herein can be prepared by per se knownmethods for the preparation of pharmaceutically acceptable compositionsthat can be administered to subjects, such that an effective quantity ofthe active substance is combined in a mixture with a pharmaceuticallyacceptable vehicle. Suitable vehicles are described, for example, inRemington's Pharmaceutical Sciences (Remington's PharmaceuticalSciences, 20^(th) ed., Mack Publishing Company, Easton, Pa., USA, 2000).On this basis, the compositions include, albeit not exclusively,solutions of the substances in association with one or morepharmaceutically acceptable vehicles or diluents, and contained inbuffered solutions with a suitable pH and iso-osmotic with thephysiological fluids.

Pharmaceutical compositions include, without limitation, lyophilizedpowders or aqueous or non-aqueous sterile injectable solutions orsuspensions, which may further contain antioxidants, buffers,bacteriostats and solutes that render the compositions substantiallycompatible with the tissues or the blood of an intended recipient. Othercomponents that may be present in such compositions include water,surfactants (such as Tween), alcohols, polyols, glycerin and vegetableoils, for example. Extemporaneous injection solutions and suspensionsmay be prepared from sterile powders, granules, tablets, or concentratedsolutions or suspensions. Immunoconjugate may be supplied, for examplebut not by way of limitation, as a lyophilized powder which isreconstituted with sterile water or saline prior to administration tothe patient.

Pharmaceutical compositions of the invention may comprise apharmaceutically acceptable carrier. Suitable pharmaceuticallyacceptable carriers include essentially chemically inert and nontoxiccompositions that do not interfere with the effectiveness of thebiological activity of the pharmaceutical composition. Examples ofsuitable pharmaceutical carriers include, but are not limited to, water,saline solutions, glycerol solutions, ethanol,N-(1(2,3-dioleyloxy)propyl)N,N, N-trimethylammonium chloride (DOTMA),diolesylphosphotidyl-ethanolamine (DOPE), and liposomes. Suchcompositions should contain a therapeutically effective amount of thecompound, together with a suitable amount of carrier so as to providethe form for direct administration to the patient.

The composition may be in the form of a pharmaceutically acceptable saltwhich includes, without limitation, those formed with free amino groupssuch as those derived from hydrochloric, phosphoric, acetic, oxalic,tartaric acids, etc., and those formed with free carboxyl groups such asthose derived from sodium, potassium, ammonium, calcium, ferrichydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol,histidine, procaine, etc.

In various embodiments of the invention, the pharmaceutical compositionis directly administered systemically or directly to the area of thetumor(s).

The pharmaceutical compositions may be used in methods for treatinganimals, including mammals, preferably humans, with cancer. The dosageand type of immunoconjugate to be administered will depend on a varietyof factors which may be readily monitored in human subjects. Suchfactors include the etiology and severity (grade and stage) of thecancer.

Clinical outcomes of cancer treatments using the immunoconjugates of theinvention are readily discernable by one of skill in the relevant art,such as a physician. For example, standard medical tests to measureclinical markers of cancer may be strong indicators of the treatment'sefficacy. Such tests may include, without limitation, physicalexamination, performance scales, disease markers, 12-lead ECG, tumormeasurements, tissue biopsy, cytoscopy, cytology, longest diameter oftumor calculations, radiography, digital imaging of the tumor, vitalsigns, weight, recordation of adverse events, assessment of infectiousepisodes, assessment of concomitant medications, pain assessment, bloodor serum chemistry, urinalysis, CT scan, and pharmacokinetic analysis.Furthermore, synergistic effects of a combination therapy comprising theimmunoconjugate and another cancer therapeutic may be determined bycomparative studies with patients undergoing monotherapy.

Another embodiment of the invention is a kit for treating or preventingcancer comprising an effective amount of the immunoconjugate of theinvention, and directions for the use thereof to treat the cancer.

In the majority of approved anticancer therapies, the anticancer therapyis used in combination with other anticancer therapies. Accordingly, theinvention provides a method of preventing or treating cancer using theimmunoconjugate of the invention in combination with at least oneadditional anticancer therapy. The other cancer therapy may beadministered prior to, overlapping with, concurrently, and/or afteradministration of the immunoconjugate. When administered concurrently,the immunoconjugate and the other cancer therapeutic may be administeredin a single formulation or in separate formulations, and if separately,then optionally, by different modes of administration. The combinationof one or more immunoconjugates and one or more other cancer therapiesmay synergistically act to combat the tumor or cancer. The other cancertherapies include, without limitation, radiation and other anticancertherapeutic agents. These other cancer therapeutics may include, withoutlimitation, 2,2′,2″trichlorotriethylamine, 6-azauridine,6-diazo-5-oxo-L-norleucine, 6-mercaptopurine, aceglarone, aclacinomycinsactinomycin, altretamine, aminoglutethimide, aminoglutethimide,amsacrine, anastrozole, ancitabine, angiogenin antisenseoligonucleotide, anthramycin, azacitidine, azaserine, aziridine,batimastar, bcl-2 antisense oligonucleotide, benzodepa, bicalutamide,bisantrene, bleomycin, buserelin, busulfan, cactinomycin, calusterone,carboplatin, carboquone, caminomycin, carmofur, carmustine, carubicin,carzinophilin, chlorambucil, chlornaphazine, chiormadinone acetate,chlorozotocin, chromomycins, cisplatin, cladribine, cyclophosphamide,cytarabine, dacarbazine, dactinomycin, daunorubicin, defosfamide,demecolcine, denopterin, detorubicin, diaziquone, docetaxel,doxifluridine, doxorubicin, droloxifene, dromostanolone, edatrexate,eflomithine, elliptinium acetate, emitefur, enocitabune, epirubicin,epitiostanol, esorubicin, estramustine, etoglucid, etoposide, fadrozole,fenretinide, floxuridine, fludarabine, fluorouracil, flutamide, folinicacid, formestane, fosfestrol, fotemustine, gallium nitrate, gemcitabine,goserelin, hexestrol, hydroxyurea, idarubicin, ifosfamide, improsulfan,interferon-alpha, interferon-beta, interferon-gamma, interleukin-2,L-asparaginase, lentinan, letrozole, leuprolide, lomustine, lonidamine,mannomustine, marcellomycin, mechlorethamine, mechlorethamine oxidehydrochloride, medroxyprogesterone, megestrol acetate, melengestrol,melphalan, menogaril, mepitiostane, methotrexate, meturedepa,miboplatin, miltefosine, mitobronitol, mitoguazone, mitolactol,mitomycins, mitotane, mitoxantrone, mopidamol, mycophenolic acid,nilutamide, nimustine, nitracine, nogalamycin, novembichin, olivomycins,oxaliplatin, paclitaxel, pentostatin, peplomycin, perfosfamide,phenamet, phenesterine, pipobroman, piposulfan, pirarubicin, piritrexim,plicamycin, podophyllinic acid 2-ethyl-hydrazide, polyestradiolphosphate, porfimer sodium, porfiromycin, prednimustine, procabazine,propagermanium, PSK, pteropterin, puromycin, quelamycin, ranimustine,razoxane, rodorubicin, roquinimex, sizofican, sobuzoxane,spirogermanium, streptonigrin, streptozocin, tamoxifen, taxotere,tegafur, temozolomide, teniposide, tenuzonic acid, testolacone,thiamiprine, thioguanine, thiotepa, Tomudex, topotecan, toremifene,triaziquone, triethylenemelamine, triethylenephosphoramide,triethylenethiophosphoramide, trilostane, trimetrexate, triptorelin,trofosfamide, trontecan, tubercidin, ubenimex, uracil mustard, uredepa,urethan, vinblastine, vincristine, zinostatin, and zorubicin, cytosinearabinoside, gemtuzumab, thioepa, cyclothosphamide, antimetabolites(e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine,5-fluorouracil, fludarabine, gemcitabine, dacarbazine, temozoamide),hexamethylmelamine, LYSODREN, nucleoside analogues, plant alkaloids(e.g., Taxol, paclitaxel, camptothecin, topotecan, irinotecan(CAMPTOSAR, CPT-11), vincristine, vinca alkyloids such as vinblastine.)podophyllotoxin, epipodophyllotoxin, VP-16 (etoposide), cytochalasin B,gramicidin D, ethidium bromide, emetine, anthracyclines (e.g.,daunorubicin), doxorubicin liposomal, dihydroxyanthracindione,mithramycin, actinomycin D, aldesleukin, allutamine, biaomycin,capecitabine, carboplain, chlorabusin, cyclarabine, daclinomycin,floxuridhe, lauprolide acetate, levamisole, lomusline, mercaptopurino,mesna, mitolanc, pegaspergase, pentoslatin, picamycin, riuxlmab,campath-1, straplozocin, tretinoin, VEGF antisense oligonucleotide,vindesine, and viriorelbine. Compositions comprising one or more cancertherapeutics (e.g., FLAG, CHOP) are also contemplated by the presentinvention. FLAG comprises fludarabine, cytosine arabinoside (Ara-C) andG-CSF. CHOP comprises cyclophosphamide, vincristine, doxorubicin, andprednisone. For a full listing of cancer therapeutics known in the art,see, e.g., the latest editions of The Merck Index and the Physician'sDesk Reference.

Pharmaceutical compositions for combination therapy may also include,without limitation, antibiotics (e.g., dactinomycin, bleomycin,mithramycin, anthramycin), asparaginase, Bacillus and Guerin, diphtheriatoxin, procaine, tetracaine, lidocaine, propranolol, anti-mitoticagents, abrin, ricinA, Pseudomonas exotoxin, nerve growth factor,platelet derived growth factor, tissue plasminogen activator,antihistaminic agents, anti-nausea agents, etc.

Indeed, administration of an effective amount of an immunoconjugate to apatient in need of such treatment may result in reduced doses of anothercancer therapeutic having clinically significant efficacy. Such efficacyof the reduced dose of the other cancer therapeutic may not be observedabsent administration with an immunoconjugate. Accordingly, the presentinvention provides methods for treating a tumor or cancer comprisingadministering a reduced dose of one or more other cancer therapeutics.

Moreover, combination therapy comprising an immunoconjugate to a patientin need of such treatment may permit relatively short treatment timeswhen compared to the duration or number of cycles of standard treatmentregimens. Accordingly, the present invention provides methods fortreating a tumor or cancer comprising administering one or more othercancer therapeutics for relatively short duration and/or in fewertreatment cycles.

Thus, in accordance with the present invention, combination therapiescomprising an immunoconjugate and another cancer therapeutic may reducetoxicity (i.e., side effects) of the overall cancer treatment. Forexample, reduced toxicity, when compared to a monotherapy or anothercombination therapy, may be observed when delivering a reduced dose ofimmunoconjugate and/or other cancer therapeutic, and/or when reducingthe duration of a cycle (i.e., the period of a single administration orthe period of a series of such administrations), and/or when reducingthe number of cycles.

Accordingly, the invention provides a pharmaceutical compositioncomprising an immunoconjugate and one or more additional anticancertherapeutic, optionally in a pharmaceutically acceptable carrier.

The present invention also provides a kit comprising an effective amountof an immunoconjugate, optionally, in combination with one or more othercancer therapeutic, together with instructions for the use thereof totreat cancer.

As stated above, combination therapy with an immunoconjugate maysensitize the cancer or tumor to administration of an additional cancertherapeutic. Accordingly, the present invention contemplates combinationtherapies for preventing, treating, and/or preventing recurrence ofcancer comprising administering an effective amount of animmunoconjugate prior to, subsequently, or concurrently with a reduceddose of a cancer therapeutic. For example, initial treatment with animmunoconjugate may increase the sensitivity of a cancer or tumor tosubsequent challenge with a dose of cancer therapeutic. This dose isnear, or below, the low range of standard dosages when the cancertherapeutic is administered alone, or in the absence of animmunoconjugate. When concurrently administered, the immunoconjugate maybe administered separately from the cancer therapeutic, and optionally,via a different mode of administration.

In an alternate embodiment, administration of the additional cancertherapeutic may sensitize the cancer or tumor to the immunoconjugate orbinding protein. In such an embodiment, the additional cancertherapeutic may be given prior to administration of the immunoconjugateor binding protein.

In one embodiment, the additional cancer therapeutic comprisescisplatin, e.g., PLATINOL or PLATINOL-AQ (Bristol Myers), at a doseranging from approximately 5 to 10, 11 to 20, 21 to 40, or 41 to 75mg/m²/cycle.

In another embodiment, the additional cancer therapeutic comprisescarboplatin, e.g., PARAPLATIN (Bristol Myers), at a dose ranging fromapproximately 2 to 3, 4 to 8, 9 to 16, 17 to 35, or 36 to 75mg/m²/cycle.

In another embodiment, the additional cancer therapeutic comprisescyclophosphamide, e.g., CYTOXAN (Bristol Myers Squibb), at a doseranging from approximately 0.25 to 0.5, 0.6 to 0.9, 1 to 2, 3 to 5, 6 to10, 11 to 20, or 21 to 40 mg/kg/cycle.

In another embodiment, the additional cancer therapeutic comprisescytarabine, e.g., CYTOSAR-U (Pharmacia & Upjohn), at a dose ranging fromapproximately 0.5 to 1, 2 to 4, 5 to 10, 11 to 25, 26 to 50, or 51 to100 mg/m²/cycle. In another embodiment, the additional cancertherapeutic comprises cytarabine liposome, e.g., DEPOCYT (Chiron Corp.),at a dose ranging from approximately 5 to 50 mg/m²/cycle.

In another embodiment, the additional cancer therapeutic comprisesdacarbazine, e.g., DTIC or DTICDOME (Bayer Corp.), at a dose rangingfrom approximately 15 to 250 mg/m²/cycle or ranging from approximately0.2 to 2 mg/kg/cycle.

In another embodiment, the additional cancer therapeutic comprisestopotecan, e.g., HYCAMTIN (SmithKline Beecham), at a dose ranging fromapproximately 0.1 to 0.2, 0.3 to 0.4, 0.5 to 0.8, or 0.9 to 1.5mg/m²/Cycle.

In another embodiment, the additional cancer therapeutic comprisesirinotecan, e.g., CAMPTOSAR (Pharmacia & Upjohn), at a dose ranging fromapproximately 5 to 9, 10 to 25, or 26 to 50 mg/m²/cycle.

In another embodiment, the additional cancer therapeutic comprisesfludarabine, e.g., FLUDARA (Berlex Laboratories), at a dose ranging fromapproximately 2.5 to 5, 6 to 10, 11 to 15, or 16 to 25 mg/m²/cycle.

In another embodiment, the additional cancer therapeutic comprisescytosine arabinoside (Ara-C) at a dose ranging from approximately 200 to2000 mg/m²/cycle, 300 to 1000 mg/m²/cycle, 400 to 800 mg/m²/cycle, or500 to 700 mg/m²/cycle.

In another embodiment, the additional cancer therapeutic comprisesdocetaxel, e.g., TAXOTERE (Rhone Poulenc Rorer) at a dose ranging fromapproximately 6 to 10, 11 to 30, or 31 to 60 mg/m²/cycle.

In another embodiment, the additional cancer therapeutic comprisespaclitaxel, e.g., TAXOL (Bristol Myers Squibb), at a dose ranging fromapproximately 10 to 20, 21 to 40, 41 to 70, or 71 to 135 mg/kg/cycle.

In another embodiment, the additional cancer therapeutic comprises5-fluorouracil at a dose ranging from approximately 0.5 to 5mg/kg/cycle, 1 to 4 mg/kg/cycle, or 2-3 mg/kg/cycle.

In another embodiment, the additional cancer therapeutic comprisesdoxorubicin, e.g., ADRIAMYCIN (Pharmacia & Upjohn), DOXIL (Alza), RUBEX(Bristol Myers Squibb), at a dose ranging from approximately 2 to 4, 5to 8, 9 to 15, 16 to 30, or 31 to 60 mg/kg/cycle.

In another embodiment, the additional cancer therapeutic comprisesetoposide, e.g., VEPESID (Pharmacia & Upjohn), at a dose ranging fromapproximately 3.5 to 7, 8 to 15, 16 to 25, or 26 to 50 mg/m²/cycle.

In another embodiment, the additional cancer therapeutic comprisesvinblastine, e.g., VELBAN (Eli Lilly), at a dose ranging fromapproximately 0.3 to 0.5, 0.6 to 0.9, 1 to 2, or 3 to 3.6 mg/m²/cycle.

In another embodiment, the additional cancer therapeutic comprisesvincristine, e.g., ONCOVIN (Eli Lilly), at a dose ranging fromapproximately 0.1, 0.2, 0.3, 0.4, 0.5, 0.6 or 0.7 mg/m²/cycle.

In another embodiment, the additional cancer therapeutic comprisesmethotrexate at a dose ranging from approximately 0.2 to 0.9, 1 to 5, 6to 10, or 11 to 20 mg/m²/cycle.

In another embodiment, an immunoconjugate is administered in combinationwith at least one other immunotherapeutic which includes, withoutlimitation, rituxan, rituximab, campath-1, gemtuzumab, and trastuzutmab.

In another embodiment, an immunoconjugate is administered in combinationwith one or more anti-angiogenic agents which include, withoutlimitation, angiostatin, thalidomide, kringle 5, endostatin, Serpin(Serine Protease Inhibitor), anti-thrombin, 29 kDa N-terminal and a 40kDa C-terminal proteolytic fragments of fibronectin, 16 kDa proteolyticfragment of prolactin, 7.8 kDa proteolytic fragment of plateletfactor-4, a 13 amino acid peptide corresponding to a fragment ofplatelet factor-4 (Maione et al., 1990, Cancer Res. 51:2077-2083), a14-amino acid peptide corresponding to a fragment of collagen I (Tolmaet al., 1993, J. Cell Biol. 122:497-511), a 19 amino acid peptidecorresponding to a fragment of Thrombospondin I (Tolsma et al., 1993, J.Cell Biol. 122:497-511), a 20-amino acid peptide corresponding to afragment of SPARC (Sage et al., 1995, J. Cell. Biochem. 57:1329-1334),and a variant thereof, including a pharmaceutically acceptable saltthereof.

In another embodiment, an immunoconjugate is administered in combinationwith a regimen of radiation therapy. The therapy may also comprisesurgery and/or chemotherapy. For example, the immunoconjugate may beadministered in combination with radiation therapy and cisplatin(Platinol), fluorouracil (5-FU, Adrucil), carboplatin (Paraplatin),and/or paclitaxel (Taxol). Treatment with the immunoconjugate may allowuse of lower doses of radiation and/or less frequent radiationtreatments, which may for example, reduce the incidence of severe sorethroat that impedes swallowing function potentially resulting inundesired weight loss or dehydration.

In another embodiment, an immunoconjugate is administered in combinationwith one or more cytokines which include, without limitation, alymphokine, tumor necrosis factors, tumor necrosis factor-like cytokine,lymphotoxin, interferon, macrophage inflammatory protein, granulocytemonocyte colony stimulating factor, interleukin (including, withoutlimitation, interleukin-1, interleukin-2, interleukin-6, interleukin-12,interleukin-15, interleukin-18), and a variant thereof, including apharmaceutically acceptable salt thereof.

In yet another embodiment, an immunoconjugate is administered incombination with a cancer vaccine or biological agents including,without limitation, autologous cells or tissues, non-autologous cells ortissues, carcinoembryonic antigen, alpha-fetoprotein, human chorionicgonadotropin, BCG live vaccine, Mycobacterial cell wall-DNA complexes,melanocyte lineage proteins, and mutated, tumor-specific antigens.

In yet another embodiment, an immunoconjugate is administered inassociation with hormonal therapy. Hormonal therapeutics include,without limitation, a hormonal agonist, hormonal antagonist (e.g.,flutamide, tamoxifen, leuprolide acetate (LUPRON)), and steroid (e.g.,dexamethasone, retinoid, betamethasone, cortisol, cortisone, prednisone,dehydrotestosterone, glucocorticoid, mineralocorticoid, estrogen,testosterone, progestin).

In yet another embodiment, an immunoconjugate is administered inassociation with a gene therapy program to treat or prevent cancer.

Combination therapy may thus increase the sensitivity of the cancer ortumor to the administered immunoconjugate and/or additional cancertherapeutic. In this manner, shorter treatment cycles may be possiblethereby reducing toxic events. The cycle duration may vary according tothe specific cancer therapeutic in use. The invention also contemplatescontinuous or discontinuous administration, or daily doses divided intoseveral partial administrations. An appropriate cycle duration for aspecific cancer therapeutic will be appreciated by the skilled artisan,and the invention contemplates the continued assessment of optimaltreatment schedules for each cancer therapeutic. Specific guidelines forthe skilled artisan are known in the art. See, e.g., Therasse et al.,2000, “New guidelines to evaluate the response to treatment in solidtumors. European Organization for Research and Treatment of Cancer,National Cancer Institute of the United States, National CancerInstitute of Canada,” J Natl Cancer Inst. February 2; 92(3):205-16.

It is contemplated that the immunoconjugate may be administered by anysuitable method such as injection, oral administration, inhalation,transdermal or intratumorally, whereas any other cancer therapeutic maybe delivered to the patient by the same or another mode ofadministration. Additionally, where multiple cancer therapeutics areintended to be delivered to a patient, the immunoconjugate and one ormore of the other cancer therapeutics may be delivered by one method,whereas other cancer therapeutics may be delivered by another mode ofadministration.

(F) Diagnostic Methods and Agents Using the Binding Proteins andImmunotoxins of the Invention

The binding proteins of the invention bind selectively to cancer cellsor molecules internalized by cancer cells, and not significantly tonormal cells. Therefore the binding proteins can be used in thediagnosis of cancer. As stated above, the inventors have shown that thebinding proteins of the invention bind to glucose transporter 8 orvariants thereof; proteins comprising any one of the amino acid sequencedefined by SEQ ID NOS:11-20; or a cancer-associated variant of glucosetransporter 8. In one embodiment of the invention, the cancer-associatedvariant of GLUT8, comprises the amino acid sequence defined by any oneof SEQ ID NOS: 11, 12 or 13, or variants thereof. In another embodimentof the invention, the cancer-associated variant of GLUT8, comprisesGLUT8 that has a modification in the N-terminal di-leucine motif. In afurther embodiment of the invention, the N-terminal di-leucine motif hasbeen modified to di-alanine.

In a preferred embodiment, the binding proteins are antibodies orantibody fragments of the invention. In addition, cancer cells may beevaluated to determine their susceptibility to the treatment methods ofthe invention by, for example, obtaining a sample of the cancer cellsand determining the ability of the sample to bind to the bindingproteins of the invention, preferably antibodies or antibody fragments.

Accordingly, the present invention includes diagnostic methods, agents,and kits that can be used by themselves or prior to, during orsubsequent to the therapeutic method of the invention in order todetermine whether or not cancer cells are present that express theantigen and can bind to the binding proteins of the invention,preferably antibodies and antibody fragments.

In one embodiment, the invention provides a method of detecting ormonitoring cancer in a subject comprising the steps of

-   -   (1) contacting a test sample taken from said subject with the        binding proteins of the invention and that binds specifically to        an antigen on the cancer cell to produce a binding        protein-antigen complex;    -   (2) measuring the amount of binding protein-antigen complex in        the test sample; and    -   (3) comparing the amount of binding protein-antigen complex in        the test sample to a control.

In one embodiment, the antigen is glucose transporter 8 or a variantthereof; a protein comprising any one of the amino acid sequencesdefined by SEQ ID NOS: 11-20, preferably SEQ ID NOS:11, 12 or 13; or acancer-associated variant of glucose-transporter 8. In one embodiment,the cancer-associated variant of glucose transporter 8 comprises amutation in the N-terminal di-leucine motif such that the variantglucose transporter 8 is localized in the cell membrane.

The invention further includes a kit for diagnosing cancer comprisingany one of the binding proteins of the invention that binds to anantigen on the cancer cell and instructions for the use thereof todiagnose the cancer.

For use in the diagnostic applications, the binding proteins of theinvention, preferably antibodies or antibody fragments, may be labeledwith a detectable marker such as a radio-opaque or radioisotope, such as³H, ¹⁴C, ³²P, ³⁵S, ¹²³I, ¹²⁵I, ¹³¹I; a fluorescent (fluorophore) orchemiluminescent (chromophore) compound, such as fluoresceinisothiocyanate, rhodamine or luciferin; an enzyme, such as alkalinephosphatase, beta-galactosidase or horseradish peroxidase; an imagingagent; or a metal ion. As described above, methods of attaching a labelto a binding protein, such as an antibody or antibody fragment, areknown in the art.

Another aspect of the invention is a method of detecting or monitoringcancer in a subject comprising the steps of

(1) measuring the amount of antibodies of the invention in a test sampletaken from said subject; and

(2) comparing the amount of antibodies of the invention in the testsample to a control.

In one embodiment, the amount of antibodies of the invention is measuredby measuring the amount of antibodies of the invention in the testsample, for example by ELISA. In another embodiment, the amount ofantibodies of the invention is measured by measuring the expressionlevels of nucleic acids encoding the antibodies of the invention in thetest sample, for example by RT-PCR.

(G) Pharmaceutical Compositions, Methods and Uses of the NovelCancer-Associated Antigen

The invention provides a novel cancer-associated antigen that isexpressed on the surface of cancer cells and not significantly expressedon the surface of normal cells. Thus, the novel cancer-associatedantigen can be used in therapies to treat and prevent cancer, includingusing the novel cancer-associated antigen or fragments thereof to elicitan immune response in vivo. In addition, the invention includes usingthe novel cancer-associated variant of GLUT8 to detect or monitorcancer.

(i) Pharmaceutical Compositions

One embodiment of the invention is a pharmaceutical compositioncomprising an effective amount of the novel-cancer associated variant ofGLUT8 or fragment thereof in admixture with a suitable diluent orcarrier. Another embodiment of the invention is a pharmaceuticalcomposition comprising an effective amount of an isolated nucleic acidencoding the novel cancer-associated variant of GLUT8 or a fragmentthereof in admixture with a suitable diluent or carrier. A furtheraspect of the invention is a pharmaceutical composition comprising aneffective amount of a recombinant expression comprising an nucleic acidsequence encoding the novel cancer-associated variant of GLUT8 or afragment thereof in admixture with a suitable diluent or carrier.

For example, the pharmaceutical compositions of the invention can beused to treat or prevent cancer. In addition, the pharmaceuticalcompositions can be used to elicit an immune response in a subjectagainst the novel cancer-associated variant of GLUT8.

The pharmaceutical composition can be prepared and administered asdiscussed above. The pharmaceutical composition can be used incombination with other anti-cancer therapeutic agents as discussedabove.

Immunogencicity can be significantly improved if the immunizing agents(i.e. the novel cancer-associated variant of GLUT8 or fragment thereof,and/or nucleic acid sequences coding thereof, and/or recombinantexpression vectors) and/or composition is, regardless of administrationformat, co-immunized with an adjuvant. Commonly, adjuvants are used as a0.05 to 1.0 percent solution in phosphate buffered saline. Adjuvantsenhance the immunogencity of an immunogen but are not necessarilyimmunogenic in of themselves. Adjuvants may act by retaining theimmunogen locally near the site of administration to produce a depoteffect facilitating a slow, sustained release of immunogen to cells ofthe immune system. Adjuvants can also attract cells of the immune systemto an immunogen depot and stimulate such cells to elicit immuneresponse. As such, embodiments of this invention encompasspharmaceutical compositions further comprising adjuvants.

Adjuvants have been used for many years to improve the host immuneresponses to, for example, vaccines. Intrinsic adjuvants (such aslipopolysaccharides) normally are the components of killed or attenuatedbacteria used as vaccines. Extrinsic adjuvants are immunomodulatorswhich are typically non-covalently linked to antigens and are formulatedto enhance the host immune responses. Thus, adjuvants have beenidentified that enhance the immune response to antigens deliveredparenterally. Some of these adjuvants are toxic, however, and can causeundesirable side-effects making them unsuitable for use in humans andmany animals. Indeed, only aluminum hydroxide and aluminum phosphate(collectively commonly referred to as alum) are routinely used asadjuvants in human and veterinary vaccines. The efficacy of alum inincreasing antibody responses to diphtheria and tetanus toxoids is wellestablished. Notwithstanding, it does have limitations. For example,alum is ineffective for influenza vaccination and inconsistently elicitsa cell mediated immune response with other immunogens. The antibodieselicited by alum-adjuvanted antigens are mainly of the IgG1 isotype inthe mouse, which may not be optimal for protection by some vaccinalagents.

A wide range of extrinsic adjuvants can provoke potent immune responsesto immunogens. These include saponins complexed to membrane proteinantigens (immune stimulating complexes), pluronic polymers with mineraloil, killed mycobacteria and mineral oil, Freund's complete adjuvant,bacterial products such as muramyl dipeptide (MDP) andlipopolysaccharide (LPS), as well as lipid A, and liposomes.

In one aspect of this invention, adjuvants useful in any of theembodiments of the invention described herein are as follows. Adjuvantsfor parenteral immunization include aluminum compounds (such as aluminumhydroxide, aluminum phosphate, and aluminum hydroxy phosphate). Theantigen can be precipitated with, or adsorbed onto, the aluminumcompound according to standard protocols. Other adjuvants such as RIBI(ImmunoChem, Hamilton, Mont.) can also be used in parenteraladministration.

Adjuvants for mucosal immunization include bacterial toxins (e.g., thecholera toxin (CT), the E. coli heat-labile toxin (LT), the Clostridiumdifficile toxin A and the pertussis toxin (PT), or combinations,subunits, toxoids, or mutants thereof). For example, a purifiedpreparation of native cholera toxin subunit B (CTB) can be of use.Fragments, homologs, derivatives, and fusion to any of these toxins arealso suitable, provided that they retain adjuvant activity. Preferably,a mutant having reduced toxicity is used. Suitable mutants have beendescribed (e.g., in WO 95/17211 (Arg-7-Lys CT mutant), WO 96/6627(Arg-192-Gly LT mutant), and WO 95/34323 (Arg-9-Lys and Glu-129-Gly PTmutant)). Additional LT mutants that can be used in the methods andcompositions of the invention include, for example Ser-63-Lys,Ala-69-Gly, Glu-110-Asp, and Glu-112-Asp mutants. Other adjuvants (suchas a bacterial monophosphoryl lipid A (MPLA) of various sources (e.g.,E. coli, Salmonella minnesota, Salmonella typhimurium, or Shigellaflexneri, saponins, or polylactide glycolide (PLGA) microspheres) canalso be used in mucosal administration.

Adjuvants useful for both mucosal and parenteral immunization includepolyphosphazene (for example, WO 95/2415), DC-chol (3b-(N—(N′,N′-dimethyl aminomethane)-carbamoyl) cholesterol (for example,U.S. Pat. No. 5,283,185 and WO 96/14831) and QS-21 (for example, WO88/9336).

A subject may be immunized with a pharmaceutical composition comprisingthe cancer-associated variant of GLUT8 or fragments thereof, an isolatednucleic acid sequence encoding thereof and/or a recombinant expressionvectors by any conventional route as is known to one skilled in the art.This may include, for example, immunization via a mucosal (e.g., ocular,intranasal, oral, gastric, pulmonary, intestinal, rectal, vaginal, orurinary tract) surface, via the parenteral (e.g., subcutaneous,intradermal, intramuscular, intravenous, or intraperitoneal) route orintranodally. Preferred routes depend upon the choice of the immunogenas will be apparent to one skilled in the art. The administration can beachieved in a single dose or repeated at intervals. The appropriatedosage depends on various parameters understood by skilled artisans suchas the immunogen itself (i.e. peptide vs. nucleic acid (and morespecifically type thereof)), the route of administration and thecondition of the animal to be vaccinated (weight, age and the like).

The invention also provides kits comprising an effective amount of apharmaceutical composition of the invention, optionally, in combinationwith one or more other cancer therapeutic, together with instructionsfor the use thereof.

(ii) Therapeutic Methods

As mentioned above, the novel cancer-associated variant of GLUT8 ispresent on cancer cells, but not significantly on normal cells. Thus,the novel cancer-associated antigen can be used in therapeutic methodsto prevent or treat cancer. In addition, the novel cancer-associatedantigen can be used to elicit an immune response in a subject, forexample in a vaccine.

One embodiment of the invention is the use of the cancer-associatedvariant of GLUT8 or fragment thereof in the manufacture of a medicamentto treat or prevent cancer. Another embodiment of the invention is theuse of the cancer-associated variant of GLUT8 or fragment thereof in themanufacture of a medicament to elicit an immune response in a subject.

The invention also includes the use of an isolated nucleic acid sequenceencoding the cancer-associated variant of GLUT8 or fragment thereof inthe manufacture of a medicament to treat or prevent cancer. In addition,the invention includes the use of an isolated nucleic acid sequenceencoding the cancer-associated variant of GLUT8 or fragment thereof inthe manufacture of a medicament to elicit an immune response in asubject.

A further embodiment of the invention is the use of the recombinantexpression vector comprising an isolated nucleic acid sequence encodingthe cancer-associated variant of GLUT8 or fragment thereof in themanufacture of a medicament to treat or prevent cancer. Also, theinvention includes the use of the recombinant expression vectorcomprising an isolated nucleic acid sequence encoding thecancer-associated variant of GLUT8 or fragment thereof in themanufacture of a medicament to elicit an immune response in a subject.

An additional embodiment of the invention is a method of treating orpreventing cancer in a subject having or suspected of having cancercomprising administering to said subject an effective amount of acancer-associated variant of GLUT8 or fragment thereof. In addition, theinvention includes a method of treating or preventing cancer in asubject having or suspected of having cancer comprising administering tosaid subject an effective amount of the an isolated nucleic acidsequence encoding the cancer-associated variant of GLUT8 or fragmentthereof. Further, the invention includes a method of treating orpreventing cancer in a subject having or suspected of having cancercomprising administering to said subject an effective amount of arecombinant expression vector comprising an isolated nucleic acidsequence encoding the cancer-associated variant of GLUT8 or fragmentthereof.

Another embodiment of the invention is a method of inducing an immuneresponse in a subject against a cancer-associated variant of GLUT8,comprising administering to said subject an effective amount of acancer-associated variant of GLUT8 or fragment thereof. In addition, theinvention includes a method of inducing an immune response in a subjectagainst the cancer-associated variant of GLUT8, comprising administeringto said subject an effective amount of an isolated nucleic acid sequenceencoding the cancer-associated variant of GLUT8 or fragment thereof.Further, the invention includes a method of inducing an immune responsein a subject against the cancer-associated variant of GLUT8 comprisingadministering to said subject an effective amount of an recombinantexpression vector comprising an isolated nucleic acid sequence encodingthe cancer-associated variant of GLUT8 or fragment thereof.

(iii) Diagnostic Methods

The novel cancer-associated variant of GLUT8 is expressed on cancercells and is not significantly expressed on normal cells, thus thedetection of the novel cancer-associated variant of GLUT8 can be used asa diagnostic method for cancer. In a preferred embodiment, thecancer-associated variant of GLUT8 comprises the amino acid sequencedefined by any one of SEQ ID NOS: 11, 12 or 13, or variants thereof. Inanother embodiment of the invention, the cancer-associated variant ofGLUT8 comprises GLUT8 that has a modification in the N-terminaldi-leucine motif. In a further embodiment of the invention, theN-terminal di-leucine motif has been modified to di-alanine.

One embodiment of the invention is a method of detecting or monitoringcancer in a subject having or suspected of having cancer, comprisingdetecting a cancer-associated variant of GLUT8 on a cell in the sample,wherein cancer is indicated, if the cancer-associated variant of GLUT8is detected on the cell.

A number of techniques can be used to detect the cancer-associatedvariant of GLUT8 on a cell. For example, the binding proteins of theinvention can be used in immunoassays to detect cell surface expressionof the cancer-associated variant of GLUT8. A person skilled in the artwill appreciate that a number of techniques can be used to detect and/orquantify cell surface expression of the cancer-associated variant ofGLUT8, including Western blots, immunoprecipitation followed bySDS-PAGE, immunocytochemistry, FACS, protein arrays, and the like.

Another aspect of the invention is a method of detecting or monitoringcancer in a subject having or suspected of having cancer, comprisingdetecting the expression of a cancer-associated variant of GLUT8 in thecell in the sample, wherein cancer is indicated, if expression of thecancer-associated variant of GLUT8 is detected in the cell. In apreferred example, an RNA expression product encoding thecancer-associated variant of GLUT8 is used to detect the expression ofthe cancer-associated variant of GLUT8 in the cell. One skilled in theart will appreciate that the RNA expression product can be detected orquantified by detecting mRNA encoding the cancer-associated variant ofGLUT8 or a fragment thereof, or oligonucleotides, cDNA, DNA, RNA, PCRproducts, synthetic DNA, synthetic RNA, or other combinations ofnaturally occurring or modified nucleotides which specifically and/orselectively hybridize to the mRNA encoding the cancer-associated variantof GLUT8 or a fragment thereof.

A number of methods can be used to detect and/or quantify RNA expressionof the cancer-associated variant of GLUT8 in a cell including RT-PCR,nuclease protection assays, such as ribonuclease protection assays andS1 nuclease assays, and Northern blots and the like.

(H) Other Methods

Glucose transporter 8 has been shown to have sugar transportingactivity. Thus, the invention includes a method of treating orpreventing cancer in a subject by modulating the activity of thecancer-associated variant of glucose transporter 8 on or in a cancercell.

In one embodiment of the invention, the method of treating or preventingcancer in a subject comprises preventing or decreasing the function ofthe cancer-associated variant of glucose transporter 8 as a transporterof sugar. In one embodiment of the invention, a binding protein of theinvention is used to prevent or decrease the function of thecancer-associated variant of glucose transporter 8 as a transporter ofsugar.

In another embodiment of the invention, a non-antibody inhibitor ofglucose transporters is used to treat or prevent cancer in a subject.

There are several known inhibitors of the glucose transporter family ofmolecules including several members of the flavonoid family. Forexample, forskolin, phloretin (a flavonoid-like compound) andcytochalasin B are know to inhibit GLUT1 and their putative bindingsites have been identified on a 3-dimensional molecular model of GLUT-1(Salas-Burgos et al., Biophys. J. 87: 2990-2999, 2004). Quercetin, aflavonol, has been shown to inhibit GLUT2-mediated glucose transport(Song et al., J. Biol. Chem. 277:15252-15260, 2002). Oestradiol and theisoflavone phytoestrogen Genistein, are also inhibitors ofGLUT1-mediated glucose transport and putative binding sites for thesemolecules have also been proposed (Afzal et al., Biochem J. 365:707-719, 2002). The glucose transporter inhibitors forskolin,dipyridamole and isobutylmethylxanthine (IBMX) bind to both GLUT1 andGLUT4 (Hellwig & Joost, Mol. Pharmacol. 40:383-389, 1991). CytochalasinB also binds GLUT4 (Wandel et al., Biochim. Biophys. Acta 1284:56-62,1996.

In addition to these known inhibitors, a person skilled in the art willappreciate that there are a number of assays known to identifyinhibitors of glucose transporters. For example, the effect ofinhibitors on a glucose transporter can be assessed by expressing theGLUT of interest, preferably glucose transporter 8, in cells such asXenopus laevis oocytes or CHO, measuring glucose uptake in the presenceor absence of the inhibitor, and determining whether the inhibitor iscompetitive or non-competitive. Once the sequence of a given GLUTisoform is known, its sensitivity to a large number of molecules can bereadily tested to identify drug candidates.

Accordingly, in the invention includes a method of treating orpreventing cancer in a subject by administering an effective amount of aglucose transporter inhibitor to a subject in need thereof. Theinhibitors include members of the Flavonoid family, such as quercetin orgenistein, Flavonoid-like molecules, such as phloretin, Oestrogeniccompounds, including oestradiol or genistein, Forskolin, Cytochalasin B,Dipyridamole and/or Isobutylmethylxanthine (IBMX).

In another embodiment of the invention, the function of thecancer-associated variant of glucose transporter 8 is prevented ordecreased by decreasing or preventing the expression of thecancer-associated variant of glucose transporter 8 in the cell.

Standard techniques can be used to prevent or decrease the expression ofthe cancer-associated variant of glucose transporter 8 in a cellincluding using antisense, triple helix, or ribozyme molecules reactiveto the transcripts of the cancer-associated variant of glucosetransporter 8 gene.

For example, standard techniques can be utilized for the production ofantisense nucleic acid molecules, i.e., molecules which arecomplementary to a sense nucleic acid encoding a polypeptide ofinterest, e.g., complementary to the coding strand of a double-strandedcDNA molecule or complementary to an mRNA sequence. Accordingly, anantisense nucleic acid can hydrogen bond to a sense nucleic acid. Theantisense nucleic acid can be complementary to an entire coding strand,or to only a portion thereof, e.g., all or part of the protein codingregion (or open reading frame). An antisense nucleic acid molecule canbe antisense to all or part of a non-coding region of the coding strandof a nucleotide sequence encoding a polypeptide of interest. Thenon-coding regions (“5′ and 3′ untranslated regions”) are the 5′ and 3′sequences that flank the coding region and are not translated into aminoacids.

An antisense oligonucleotide can be, for example, about 5, 10, 15, 20,25, 30, 35, 40, 45 or 50 nucleotides or more in length. An antisensenucleic acid of the invention can be constructed using chemicalsynthesis and enzymatic ligation reactions using procedures known in theart. For example, an antisense nucleic acid (e.g., an antisenseoligonucleotide) can be chemically synthesized using naturally occurringnucleotides or variously modified nucleotides designed to increase thebiological stability of the molecules or to increase the physicalstability of the duplex formed between the antisense and sense nucleicacids, e.g., phosphorothioate derivatives and acridine substitutednucleotides can be used. Examples of modified nucleotides which can beused to generate the antisense nucleic acid include 5-fluorouracil,5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine,4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil,5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N-6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil,(acp3)_(w), and 2,6-diaminopurine. Alternatively, the antisense nucleicacid can be produced biologically using an expression vector into whicha nucleic acid has been subcloned in an antisense orientation (i.e., RNAtranscribed from the inserted nucleic acid will be of an antisenseorientation to a target nucleic acid of interest).

Antisense nucleic acid molecules administered to a subject or generatedin situ such that they hybridize with or bind to cellular mRNA encodingthe polypeptide of interest to thereby inhibit expression, e.g., byinhibiting transcription and/or translation. The hybridization can be byconventional nucleotide complementarity to form a stable duplex, or, forexample, in the case of an antisense nucleic acid molecule which bindsto DNA duplexes, through specific interactions in the major groove ofthe double helix. An example of a route of administration of antisensenucleic acid molecules of the invention includes direct injection at atissue site. Alternatively, antisense nucleic acid molecules can bemodified to target selected cells and then administered systemically.For example, for systemic administration, antisense molecules can bemodified such that they specifically bind to receptors or antigensexpressed on a selected cell, e.g., a T cell or brain cell, e.g., bylinking the antisense nucleic acid molecules to peptides or antibodieswhich bind to cell surface receptors or antigens. The antisense nucleicacid molecules can also be delivered to cells using vectors, e.g., genetherapy vectors, described below. To achieve sufficient intracellularconcentrations of the antisense molecules, vector constructs in whichthe antisense nucleic acid molecule is placed under the control of astrong pol II or pol III promoter are preferred.

An antisense nucleic acid molecule of interest can be an α-anomericnucleic acid molecule. An α-anomeric nucleic acid molecule formsspecific double-stranded hybrids with complementary RNA in which,contrary to the usual α-units, the strands run parallel to each other(Gaultier et al., 1987, Nucleic Acids Res. 15:6625-6641). The antisensenucleic acid molecule can also comprise a 2′-o-methylribonucleotide(Inoue et al., 1987, Nucleic Acids Res. 15:6131-6148) or a chimericRNA-DNA analogue (Inoue et al., 1987, FEBS Lett. 215:327-330).

Ribozymes are catalytic RNA molecules with ribonuclease activity thatare capable of cleaving a single-stranded nucleic acid, such as an mRNA,to which they have a complementary region, and can also be generatedusing standard techniques. Thus, ribozymes (e.g., hammerhead ribozymes(described in Haselhoff and Gerlach, 1988, Nature 334:585-591)) can beused to catalytically cleave mRNA transcripts to thereby inhibittranslation of the protein encoded by the mRNA. A ribozyme havingspecificity for a nucleic acid molecule encoding a polypeptide ofinterest can be designed based upon the nucleotide sequence of a cDNAencoding a cancer-associated variant of GLUT8. For example, a derivativeof a Tetrahymena L-19 IVS RNA can be constructed in which the nucleotidesequence of the active site is complementary to the nucleotide sequenceto be cleaved in a Cech et al. U.S. Pat. No. 4,987,071; and Cech et al.U.S. Pat. No. 5,116,742. Alternatively, an mRNA encoding a polypeptideof interest can be used to select a catalytic RNA having a specificribonuclease activity from a pool of RNA molecules. See, e.g., Barteland Szostak, 1993, Science 261:1411-1418.

Triple helical structures can also be generated using well knowntechniques. For example, expression of a polypeptide of interest can beinhibited by targeting nucleotide sequences complementary to theregulatory region of the gene encoding the polypeptide (e.g., thepromoter and/or enhancer) to form triple helical structures that preventtranscription of the gene in target cells. See generally Helene, 1991,Anticancer Drug Des. 6(6):569-84; Helene, 1992, Ann. N.Y. Acad. Sci.660:27-36; and Maher, 1992, Bioassays 14(12):807-15.

In various embodiments, nucleic acid compositions can be modified at thebase moiety, sugar moiety or phosphate backbone to improve, e.g., thestability, hybridization, or solubility of the molecule. For example,the deoxyribose phosphate backbone of the nucleic acids can be modifiedto generate peptide nucleic acids (see Hyrup et al., 1996, Bioorganic &Medicinal Chemistry 4(1): 5-23). As used herein, the terms “peptidenucleic acids” or “PNAs” refer to nucleic acid mimics, e.g., DNA mimics,in which the deoxyribose phosphate backbone is replaced by apseudopeptide backbone and only the four natural nucleobases areretained. The neutral backbone of PNAs has been shown to allow forspecific hybridization to DNA and RNA under conditions of low ionicstrength. The synthesis of PNA oligomers can be performed using standardsolid phase peptide synthesis protocols as described in Hyrup et al.,1996, supra; Perry-O'Keefe et al., 1996, Proc. Natl. Acad. Sci. USA 93:14670-675.

PNAs can, for example, be modified, e.g., to enhance their stability orcellular uptake, by attaching lipophilic or other helper groups to PNA,by the formation of PNA-DNA chimeras, or by the use of liposomes orother techniques of drug delivery known in the art. For example, PNA-DNAchimeras can be generated which may combine the advantageous propertiesof PNA and DNA. Such chimeras allow DNA recognition enzymes, e.g., RNAseH and DNA polymerases, to interact with the DNA portion while the PNAportion would provide high binding affinity and specificity. PNA-DNAchimeras can be linked using linkers of appropriate lengths selected interms of base stacking, number of bonds between the nucleobases, andorientation (Hyrup, 1996, supra). The synthesis of PNA-DNA chimeras canbe performed as described in Hyrup, 1996, supra, and Finn et al., 1996,Nucleic Acids Res. 24(17):3357-63. For example, a DNA chain can besynthesized on a support using standard phosphoramidite couplingchemistry and modified nucleoside analogs. Compounds such as5′-(4-methoxytrityl)amino-5′-deoxy-thymidine phosphoramidite can be usedas a link between the PNA and the 5′ end of DNA (Mag et al., 1989,Nucleic Acids Res. 17:5973-88). PNA monomers are then coupled in astepwise manner to produce a chimeric molecule with a 5′ PNA segment anda 3′ DNA segment (Finn et al., 1996, Nucleic Acids Res. 24(17):3357-63).Alternatively, chimeric molecules can be synthesized with a 5′ DNAsegment and a 3′ PNA segment (Peterser et al., 1975, Bioorganic Med.Chem. Lett. 5:1119-11124).

In other embodiments, the oligonucleotide may include other appendedgroups such as peptides (e.g., for targeting host cell receptors invivo), or agents facilitating transport across the cell membrane (see,e.g., Letsinger et al., 1989, Proc. Natl. Acad. Sci. USA 86:6553-6556;Lemaitre et al., 1987, Proc. Natl. Acad. Sci. USA 84:648-652;International Publication No. WO 88/09810) or the blood-brain barrier(see, e.g., International Publication No. WO 89/10134). In addition,oligonucleotides can be modified with hybridization-triggered cleavageagents (see, e.g., Krol et al., 1988, Bio/Techniques 6:958-976) orintercalating agents (see, e.g., Zon, 1988, Pharm. Res. 5:539-549). Tothis end, the oligonucleotide may be conjugated to another molecule,e.g., a peptide, hybridization triggered cross-linking agent, transportagent, hybridization-triggered cleavage agent, etc.

Another aspect of the invention is a method to identify compounds thatare able to modulate the expression or activity of the cancer-associatedvariant of glucose transporter 8, which can be used to prevent or treatcancer.

In one embodiment of the invention, the method for identifying acompound for ability to prevent or treat cancer comprises the steps:

(a) contacting a cell expressing a cancer-associated variant of glucosetransporter 8 with a test compound; and

(b) determining the expression or function of the cancer-associatedvariant of glucose transporter 8;

(c) comparing the expression or function of the cancer-associatedvariant of glucose transporter 8 to a control, wherein a decrease inexpression or function of the cancer-associated variant of glucosetransporter 8 as compared to the control is indicative of a compounduseful to prevent or treat cancer.

The following non-limiting examples are illustrative of the presentinvention:

EXAMPLES Example 1 Generation of VB1-050 Monoclonal Antibody

The VB1-050 monoclonal antibody was generated from pooled lymphocytes ofcancer patient samples. SHFP-1 was used as the fusion partner togenerate the monoclonal antibody. VB1-050 is an IgG1, kappa monoclonalantibody.

Example 2 Sequencing

Messenger RNA (mRNA) was isolated from hybridoma cells and first strandcomplement DNA (cDNA) was synthesized. The cDNA was then used to isolateantibody H and L chain genes by PCR. PCR primers were designed (seenote) according to the consensus framework regions of the H (Gamma) andL (Kappa) chain isotypes. The PCR products were individually cloned intothe TOPO-pCR 2.1 vector and transformed into E. coli cells. Individualclones containing the inserts in TOPO-pCR 2.1 were isolated and grown.Plasmid DNA was purified and sequenced.

Gamma Primers: (SEQ ID NO: 21) 1) 5′ TCT AAA GAA GCC CCT GGG AGC ACA GCTCAT CAC CAT G 3′ (SEQ ID NO: 22) 2) 5′ GCC CGG GGA GCG GGG GCT TGC CGGCCG TCG CAC TCA 3′ (SEQ ID NO: 23) 3) 5′ ACC ATG AGT GAG AAA AAC TGG ATTTGT GTG GCA 3′ (SEQ ID NO: 24) 4) 5′ GGA GCC GGT GAC CAG GGT TCC CTG GCCCCA 3′ (SEQ ID NO: 25) 5) 5′ CTC ACC ATG GAG TTT GGG CTG AGC TGG GTT 3′(SEQ ID NO: 26) 6) 5′ GGA GGC TGA GGA GAC GGT GAC CAG GGT TCC CTG GCC 3′Kappa Primers: (SEQ ID NO: 27) 7) 5′ GGC TCG AGA TGG ACA TGR RRD YCC HVGYKC ASC TT 3′ (SEQ ID NO: 28) 8) 5′ CCC GTC GAC CAT CAG ATG GCG GGA AGAT 3′ Note: In order to isolate as many varieties as possible using asingle primer, mixed bases are used for certain consensus primers: R = A+ G, D = A + T + G, Y = C + T, H = A + C + T, V = A + C + G, K = T + G,S = C + G, W = A + T.

Each PCR reaction comprised the following components in a 50 μL reactionvolume.

-   -   10×PCR buffer 5 μL    -   2 mM dNTPs 5 μL    -   50 mM MgCl2 2 μL    -   5′ Primer 20 pmol.    -   3′ Primer 20 pmol.    -   Taq DNA Polymerase 2.5 U    -   DNA template 50 ng

The PCR cycling conditions were: 94° C. for 1 min., 62° C. for 1 min.,72° C. for 1.5 min. for 30 cycles and a final extension for 10 min. at72° C. Amplified PCR products were electrophoretically separated on a 1%agarose gel, excised, purified using a Qiaquick gel extraction kit,cloned into the TOPO pCR 2.1 cloning vector and then DNA sequenced usingthe 373 DNA sequencer stretch (Griffin G. H. and Griffin M. A.: PCRtechnology, Current innovations. CRC Press, Boca. Raton. Fla. 3431 USA;Cloning vector pCR 2.1, Catalogue #205184. Invitrogen, Carlsbad, Calif.;Qiagen, Qiaquick gel extraction kit, Catalogue # 28706. Qiagen Inc.,Mississauga, ON; and 373 DNA Stretch. PE Applied Biosystems, MississaugaON.).

The CDR sequences for VB1-050 are shown in Table 1.

The light chain variable region and the heavy chain variable region areshown in FIGS. 1 and 2, respectively.

Example 3 Antibody Profiling by Measuring Tumor and Normal CellReactivity

VB1-050 was tested by flow cytometry for tumor and normal cellreactivity. A single panel of tumor cell lines representing fifteendifferent types of epithelial cancers was screened. The VB1-050 resultsare summarized in Table 2. Although VB1-050 had a MF value >2.0 for allindications, the strongest reactivity was observed, but not limited to,breast, melanoma, and ovarian cell lines. In comparison, reactivity withnormal tissue cell lines was generally less than that seen with thecancer cell lines. In the case of the breast and prostate cell lines theexpression of VB1-050 on average was >9-fold on the cancer cell lines.The two exceptions were the kidney and lung cell lines; however, theywere still lower than the corresponding tumor cell type. MF valueindicates the mean calculated from the sum of the mean fold increase inmedian fluorescence over the control antibody from all cell lines ineach indication. A zero value indicates no measurable reactivityrelative to the control antibody.

Example 4 Normal Tissue Microarray

VB1-050 was first tested against the flow positive tumor cell lineSKBR-3 to assess the appropriate tissue format to demonstrate membranestaining and to define the optimal conditions for staining. VB1-050demonstrated strong nuclear and/or nuclear membrane staining in allexperimental groups. Of notice, the cytospin slides showed punctatestaining on the cellular membrane, in about 30% of the intact cells. Onfrozen sections similar cellular membrane staining was detected (10% ofcells) in addition to nuclear/nuclear membrane staining (60% of cells)as well as staining in the cytoplasm (10% of cells). On fixed-cellpellets, this antibody stained the nucleus and nuclear membrane (70% ofcells), and cytoplasm (10% of cells), but very rarely stained the cellmembrane (3-5% of cells). Since fixation did not affect the antigen (asevidenced by staining of fixed cells on cytospin slides) the apparentloss of cell membrane staining in the fixed cell pellet may be due tothese cells having less surface area of the membrane as compared to thefrozen cells. The greater membrane area visible in frozen cells is aconsequence of shrinkage of the cytoplasm, as well as, by nature, athicker section using frozen cells. Alternatively, processing afterfixation (embedding, etc) may have altered the surface antigen.

Once the optimal staining conditions were identified, the antibody wastested in comparison with an isotype control (4B5) on a low density (LD)array of formalin-fixed critical normal for normal tissue reactivity.These results for VB1-050 are summarized in Table 3. No significantmembrane staining of any of the normal critical tissues was observed.Intense staining of the nucleus and/or nuclear membrane was seen withmany of the tissues. Similarly, consistent cell membrane staining wasnot seen with any of the non-critical normal tissues, except testes,which showed 30% membrane staining for ⅕ tested (Table 4).

Example 5 Tumor Tissue Microarray

In contrast to the critical and non-critical normal tissue screening,cell membrane reactivity was observed with some but not all of the tumortissues. VB1-050 was more often detected in cancers of the colon,prostate, stomach, ovary and liver. The most intense staining (2+) wasconsistently detected in the gastric carcinomas. Generally, thepercentage of cells with membrane staining varied with the indicationand the tissue samples within each indication; however, carcinomas ofthe colon did show the highest percentage of cells being stained. SeeTable 5. No staining was detected in tissue specimens from lung, rectumskin and uterine cancers.

Example 6 Assessment of VB1-050 Binding and Internalization by FlowCytometry and Confocal Microscopy

VB1-050 and two control antibodies (5E9 and MA-103) that demonstratestrong reactivity against the tumor cell line A-375 were used to assessVB1-050 for internalization. A representative experiment is shown inTable 6. VB1-050 binding results at different temperatures were notdifferent from the internalizing antibody 5E9. After 60 min at 37° C.,the membrane-bound VB1-050 disappeared from the cell surface, with a61.8% reduction in median fluorescence. Increasing the incubation timeat 37° C. was associated with a further decline in median fluorescence,but at a slower rate. By 120 min, the median fluorescence had decreasedby 69.6%. Flow histograms demonstrating cell-surface binding areillustrated in FIG. 3.

To confirm whether the cell-surface bound VB1-050 internalized intoA-375 cells or was shed from the plasma membrane, antibody-treated cellswere further evaluated by direct visualization of fluorescencedistribution and intracellular staining with the aid of laser scanningconfocal microscopy. Similarly to MA-103 and 5E9, VB1-050 incubationwith A-375 cells at 4° C. for 60 min demonstrated a circumferentialsurface distribution of fluorescence label (FIG. 4A). Warming theVB1-050 antibody bound cells to 37° C. revealed strong intracellularstaining by the internalized antibody within 60 minutes, as shown inFIG. 4B.

Example 7 Binding Affinity Status

The most important factor that influences the formation ofantibody-antigen complexes is the affinity of the antibody for itsantigen. This binding affinity is a constant property of these reactantsand is expressed as an equilibrium constant (K) that is measured as aratio of association/dissociation or KA/KD. For a given antibody, thedifference in affinities observed relates more to the dissociation (KD)rather than association (KA), thus KD was chosen as a measure ofaffinity of VB1-050.

A flow cytometric approach was used to determine antibody affinity[Benedict, C. A. et al. (1997) “Determination of the binding affinity ofan anti-CD34 single-chain antibody using a novel, flow cytometry basedassay” J. Immunol. Methods 2001, 223-31]. Briefly, A-375 cells wereincubated with a range of concentrations of VB1-050 in a sufficientamount of time to achieve equilibrium. The cells were then washed andtreated with biotin conjugated anti-human IgG secondary antibody. Thetumor cells were then analyzed by flow cytometry to detect cell boundantibody. The inverse of the determined median fluorescence was plottedas a function of the inverse of antibody concentration to determine KDby the Lineweaver-Burk method [Lineweaver, H. et al. (1934) “Thedetermination of enzyme dissociation constants” J. Am. Chem. Soc. 56,658].

The KD value of the interaction between VB1-050 and A-375 was determinedto be 4.90×10⁻⁸M.

Example 8 Engineering and Testing of a De-Bouganin Immunotoxin

1) Engineering of VB6-050

The PelB—V_(H)-PvuII insert, obtained from the digestion of thePelB—V_(H845)—C_(H)—F-de-bouganin/pSV73 plasmid with EcoRI and PvuII,was ligated into the PelB(—S)—V_(H050)—C_(H)—F-de-bouganin/psV73 vectorpre-digested with the same enzymes (previously engineered for inclusionexpression containing the PelB leader without the signal peptidesequence, PelB(—S)). 10F competent cells were transformed with theligation reaction and selected on ampicillin plate. The screening ofcolonies by mapping restriction sites determined which clones containedthe PelB—V_(H050)—C_(H)—F-de-bouganin insert.

The PelB(—S)—V_(L050)—C_(L) fragment was digested with EcoRV and XhoIand ligated into the SpeI-de-bouganin-PelB—V_(L845)—C_(L)/psV73 vectorpre-digested with the same enzymes. 10F competent cells were thentransformed with the ligation reaction and plated onto LB-agar platessupplemented with ampicillin. The screening of colonies by mappingrestriction sites determined which clones contained theSpeI-de-bouganin-PelB—V_(L050)—C_(L) insert. TheSpeI-de-bouganin-PelB—V_(L050)—C_(L) insert was then cloned into thepING3302 plasmid using the EcoRI and XhoI restrictions sites. ThePelB—V_(H050)—C_(H)—F-de-bouganin fragment was digested with EcoRI andSpeI and ligated to the SpeI-de-bouganin-PelB—V_(L050)—C_(L)/3302 vectorpre-digested with the same enzyme creating VB6-050 insert. The plasmidcontaining the VB6-050 insert was then isolated and used to transformE104 cells.

2) Small-Scale Expression Studies

Transformed E104 cells containing VB6-050 were propagated in 30 mL of TBmedia (1% inoculum) in a 250 mL shake flask at 37° C. and shaken at 225rpm for approximately 5 hours until the optical density (O.D. 600 nm)reached ˜2. At this time, the culture was induced with a finalconcentration of 0.1% L-(+) arabinose and incubated at 25° C. for 16hours. Subsequently, the supernatant was collected by centrifugation at14000 rpm for 5 minutes and analyzed by Western blot using either ananti-lambda or an anti-human kappa light chain under non-reducingconditions to confirm the presence and size of the immunotoxin.

3) Master Cell Bank Generation

To generate the MCB, a single colony from an LB-agar plate containing 25μg/mL of tetracycline was used to inoculate 5 mL 2×YT plus 25 μg/mL oftetracycline and incubated at 37° C. with constant shaking. When theOD₆₀₀ reached ˜2, 50 mL of 2×YT medium containing 25 μg/mL oftetracycline in a 250 mL shake-flask was inoculated with 1.25 mL of theseed culture and incubated at 37° C. When the (OD₆₀₀ reached 1 to 1.5,25 mL of 30% glycerol was mixed into the culture. Aliquots of 1.5 mLwere placed in cryotubes and stored at −80° C. Three independent vialswere tested for expression as described previously.

4) Fermentation and Purification

Fed batch fermentation of VB6-050 was performed in a 15 L CHEMAPfermenter using TB medium. At an OD₆₀₀ of 20 (mid-log), the culture wasinduced with a mixture of feed (50% glycerol) and inducer (200 gL-arabinose). At 30 hours post induction, the culture was harvested andcentrifuged at 8000 rpm for 30 min, then purified using CM-sepharose andChelating-sepharose columns followed by a size exclusion column.Briefly, the supernatant was concentrated and diafiltered against 20 mMsodium phosphate pH 6.9±0.1. The diafiltered concentrated supernatantwas then applied onto a CM-sepharose column equilibrated with 20 mMsodium phosphate, 25 mM NaCl pH 6.9±0.1. The column was washed with 20mM sodium phosphate, 25 mM NaCl pH 6.9±0.1. Bound VB6 Fab-de-bouganinfusion protein was subsequently eluted with 20 mM sodium phosphate, 150mM NaCl pH 7.5±0.1. The CM-Sepharose eluate was adjusted to a finalconcentration of 0.25% triton-X100 and applied to a charged Chelatingsepharose column. The Chelating-sepharose column was then washed with 3different wash buffers starting with 20 mM sodium phosphate, 150 mMNaCl, 0.25% triton-X100 pH 7.5±0.1, followed by 20 mM sodium phosphate,150 mM NaCl pH 7.5±0.1 and followed by 20 mM sodium phosphate, 150 mMNaCl, 10 mm imidazole pH 7.5±0.1. The bound VB6 Fab-de-bouganin fusionprotein was eluted with 20 mM sodium phosphate, 150 mM NaCl, 250 mMimidazole pH 7.5±0.1 and collected in 2 mL fractions. The absorbance atA₂₈₀ nm was determined for each fraction and the fractions with materialwere pooled and applied onto a size exclusion column S200 in order toobtain a purity of ˜80%. Samples at each step of the process wereanalyzed by Western blot using the anti-kappa antibody. Purity after thesize exclusion column was confirmed by colloidal blue staining.

5) Biological Activity of the VB6-050

Human melanoma A-375, human T cell Daudi, human ovarian SK-OV-3, humanpancreatic Panc-1, human breast SKBR-3 and MB-435S and human colonColo-320 cell lines were grown in their respective media as per ATCCprotocols. Cells were harvested at 30 to 40% confluency with viabilitygreater than 90%.

a) Binding Activity

Flow cytometry was used to demonstrate that purified VB6-050 retainbinding specificity using antigen-positive cell line SKBR-3, A-375 andSK-OV-3 and an antigen-negative cell line Panc-1, Colo-320 and Daudi,respectively. Binding was detected using an anti-de-bouganin antibody.Briefly, constructs to be tested were incubated with 0.45×10⁶ tumorcells for 1.5 hours on ice. After washing, cell surface bound reactivitywas detected with rabbit anti-de-bouganin ( 1/100) for an hour on ice.The cells were washed and incubated with FITC-conjugated anti-rabbit IgGfor 30 minutes on ice. Subsequently, the cells were washed, resuspendedin PBS 5% FCS containing propidium iodide for assessment of Fab bindingby flow cytometry.

b) Competition Assay

A saturation curve was generated by incubating antigen positive cellwith an increased concentration of VB6-050 ranging from 10 to 750 μg/mL.The bound Fab-de-bouganin was detected by flow cytometry as describedpreviously. The concentration of Fab-de-bouganin corresponding to thesaturation point was then incubated with antigen-positive cells inpresence of increased concentrations of parental IgG antibody. Thereduction of bound Fab-de-bouganin was measured by flow cytometry. The4B5 IgG was used as a negative control.

c) Cytotoxicity Assay

The cytotoxicity of VB6-050 was measured by an MTS assay. Briefly,antigen-positive and antigen-negative cells were seeded at 1000 cellsper well and incubated at 37° C. for 3 hours. Subsequently, varyingconcentrations of VB6-050 and de-bouganin were added to the cells andafter 5 days, the cell viability determined.

Results

1) Engineering and Small-Scale Expression of VB6-050 in pING3302Expression Vector

As a first attempt to produce Fab-de-bouganin protein, the vector wasengineered as two separate constructs, the Fd portion fused tode-bouganin and the light chain. The expression of each chain wasdirected into inclusion bodies by the deletion of the peptide signal inthe PelB leader sequence, PelB(—S). However, the lack of expression ofthe Fd-de-bouganin protein into inclusion bodies and the success of theVB6-845 soluble expression rationalized the re-engineering of VB6-050soluble Fab-de-bouganin construct. In order to minimize the time ofre-engineering and based on feasibility, restriction enzymes were usedto link the Fd-F-de-bouganin and V_(L)—C_(L) fragments to the PelBleader sequence with a peptide signal.

The analysis of the Fd and light chain of 050 showed that therestriction sites EcoRI and PvuII and EcoRV and XhoI located in theheavy and light chain, respectively, allowed the re-engineering ofVB6-050 without PCR reaction using the VB6-845 intermediate constructs.To that end, the PelB insert with the leader peptide of thePelB—V_(H845)—C_(H)—F-de-bouganin was obtained using the restrictionsites EcoRI and PvuII and ligated into thePelB(—S)—V_(H050)—C_(H)—F-de-bouganin pre-digested with the same enzyme(FIG. 5A). Similarly, the PelB(—S)—V_(L050)—C_(L)/pSV73 plasmid wasdigested with EcoRV and XhoI and the insert cloned into theSpeI-de-bouganin-PelB—V_(L845)—C_(L), vector pre-digested with EcoRV andXhoI. The insert, PelB—V_(L050)—C_(L), was subsequently inserted intothe 3302 plasmid using the EcoRI and XhoI restriction sites (FIG. 5B).The PelB—V_(H050)—C_(H)—F-de-bouganin fragment then ligated via theEcoRI-SpeI restriction sites generating the VB6-050 insert into the 3302DNA plasmid which was transformed into E104 cells (FIGS. 5C and 5D).

The Western blot analysis under non-reducing conditions of VB6-050showed that the full-length proteins are detected with the anti-Kappalight chain antibody (FIG. 6). In addition, the level of expression ofVB6-050 is similar to the VB6-845 used as a reference. Western blottingof non-induced E104 culture supernatant revealed no corresponding bandssuggesting that these proteins are specifically detected with thecorresponding antibody (FIG. 6, lane 4). In addition a similar profileof degraded products observed in the VB6-845 was also obtained with eachclone.

2) Purification of VB6-050

VB6-050 was purified from a 15 liter fermentor. Aliquots from each stepsof the purification process were analyzed on Western blot in order toassess the recovery rate of each column (FIG. 7A). The immunoblot wasincubated with an anti-human Kappa light chain. No detectable productwas observed in the permeate of the concentration and diafiltered step(FIG. 7A, lane 2 and 4, respectively). The diafiltered material, diluted1/10, was loaded on the CM-sepharose column (FIG. 7A, lane 5). Westernblot analysis showed that the CM eluate (FIG. 7A, lane 8) contains thefull-length VB6-050 and possible degraded VB6-050 fragments. Theflow-through of the nickel column, lane 9, shows that most of theVB6-050 and other products bound to the column. The Ni²⁺ eluate, lane13, was then applied on a SEC 200 size exclusion allowing the separationof the intact VB6-050 from the degraded fragments (FIG. 7A, lane 14 andFIG. 7B, lane 2).

3) Detection of VB6-050 Proteins Binding by Flow Cytometry

For VB6-050, antigen-positive and antigen-negative cell lines wereselected based on the profiling data of each antibody. The boundFab-de-bouganin was detected by flow cytometry using anti-bouganinantibody. As expected, no binding was detected by flow cytometry afterincubation with the antigen-negative cell. In contrast, boundFab-de-bouganin was detected with the antigen-positive cell lines. Inaddition, the antigen positive cell line was incubated with variousconcentrations of Fab-de-bouganin protein, ranging from 0 to 500 μg/mL,and the binding activity was determined by flow cytometry. A titrationcurve was generated (FIG. 8). The inverse of the determined medianfluorescence was plotted as a function of the inverse of antibodyconcentration to determine the K_(D) by Lineweaver-Burk method. Astraight line was generated and the K_(D) was calculated from the slopeof the curve. The dissociation constant K_(D) were determined by thefollowing equation: 1/F=1/fmax+(KD/Fmax)(1/VB6), where F=backgroundsubtracted median fluorescence and Fmax was calculated from the plot(Table 7). The saturation point for the Fab-de-bouganin was determinedfrom the saturation curve and used for the competition assay with theparental antibody. The VB6-050 saturation point, 250 μg/mL, wasincubated with antigen-positive cells in presence of increasing amountof its corresponding parental IgG ranging from 0 to 1000 μg/mL. Thebound VB6-050 was detected by flow cytometry using anti-bouganinantibody. As expected, the parental IgG competed the binding of theFab-de-bouganin proteins. The concentration of IgG required to inhibit50% of bound Fab-de-bouganin was determined to be 180 μg/mL (Table 7).

4) Cytotoxicity of VB6-050 Proteins

The negative and positive-antigen cell lines were incubated withdifferent concentrations of VB6-050 from 1 nM to 1 μM. After 5 daysincubation, the calculated IC₅₀ of VB6-050 was 400 nM (FIG. 9) (Table7). In contrast, no IC₅₀ could be determined with the antigen negativecell lines.

Conclusion

The VB1-050 IgG, selected from the Hybridomics and Immunomine™ platformwas engineered as soluble Fab-de-bouganin fusion protein which containsde-bouganin genetically linked to the V_(H)—C_(H) domain via the furincleavable linker. The data confirms that the Fab-de-bouganin formatderived of IgG is suitable for soluble expression leading to an easydownstream process. Once purified, the flow cytometry data showed thatthe profiling data of the VB6 format matched the parental IgG suggestingthe specificity and selectivity was preserved. In addition, the IgGcompeted with the VB6 fusion proteins demonstrating that both fragmentsbound to the same antigen. The calculated affinity of the VB6 format wasin the micromolar range leading to IC₅₀≧ to 280 nM.

Example 9 Antigen Identification

Preliminary Characterization of VB1-050 Ag

VB1-050 showed a 58.62% (P-value 0.008) increase in binding upondeglycosylation. This increase in the binding of the antigen observedupon deglycosylation, suggests that the glycan moiety may partially maskthe antigenic sites on the cell surface and that deglycosylation may bean essential step in the identification of the antigen.

Immunoprecipitation

Equal amounts of membrane preparations from each of the four positivecell lines, MCF-7, MDA-MB-435S, A-375, HepG2, and three negative celllines, Panc-1, Daudi and C-33A were deglycosylated with N-Glycanase andnutated with 40 μg VB1-050 and 4B5-IgG each in the presence of proteaseinhibitors with conditions mimicking in-vivo conditions. Immunecomplexes were centrifuged, washed with RIP-A lysis buffer and elutedwith 0.2M glycine pH 2.5.

Gel-Based Analysis and Western Blotting

Immunoprecipitates from all the above-mentioned cell lines weresubjected to reducing and non-reducing conditions of sample preparationand were subsequently analyzed by SDS-PAGE and Western blotting. Theresulting blots were probed with 4B5-IgG and VB1-050 simultaneously andcorresponding secondary antibody conjugated to HRP, to visualize theimmunoprecipitated proteins by chemiluminescence. A single band wasdetected at −50 kDa from VB1-050 immunoprecipitates on 1D-PAGE in allthe cell lines and 2D-PAGE did not yield any result. No bands weredetected with 4B5-IgG. Since the conventional approach did not show anydifferentially expressed antigen, an alternative method for antigenidentification was explored.

HIP-Antigen ID Using ProteomeLab™ PF-2D in Tandem with Nano-ESI-MS/MS

PF2D Fractionation of HepG2, MCF-7, Panc-1 and C-33A

The pre-fractionated VB1-050 immunoprecipitates from membranepreparations were clarified of all particulate material by high speedcentrifugation. The clear supernatant was equilibrated with Start bufferand fractionated on the chromatofocusing column in the first dimension.The peak fractions eluting at pH=7.4-7.6 was equilibrated with solvent A(0.1% TFA) in the ratio of 1:4, and fractionated on the HPRP column witha gradient of 0-100% acetonitrile containing traces of TFA.

HepG2 and MCF-7 upon fractionation on the chromatofocusing column (CF),showed a single broad peak eluting. at pH 7.4-7.6 as two fractions(constituting # B6 and B7) at 68 and 65 minutes, respectively. Asobserved in FIGS. 10A and B, HepG2 and MCF-7 membranes eluting off theHPRP column showed different separation profiles, entirely dependent onthe presence of the VB1-050 reactive antigens. Two peaks were observedto be differentially regulated in the positive cell lines, that seemedto be negligible or totally absent in the negative cell lines, Panc-1and C-33A membranes (FIGS. 10A and B). On thorough analysis of theprotein peaks present in the positive cell line (MCF-7 and HepG2), itwas shown that the peaks elute from the RP-HPLC column with retentiontimes of 15 and 18 minutes, respectively. These peaks were not observedin the antigen-negative cell lines (Panc-1 and C-33A). Instead, a singlepeak eluting slightly earlier at 12 minutes was observed in the negativecell lines.

Fractionation Analysis Using ProteoVue™/DeltaVue™ software

The chromatographic profiles obtained for the HPRP column were importedinto ProteoVue™ files to be formatted into an acceptable format for thefinal analysis on DeltaVue™. The analyses were combined for the antigenfractionation from both positive (HepG2 and MCF-7) and negative (Panc-1and C-33A) cell lines and formatted using ProteoVue® software togenerate a comprehensive membrane protein map from each of the celllines. A comparative profiling of differentially regulated proteins wasthereafter generated on the DeltaVue™ software. The chromatographicprofiles of the fractionation from both cell lines were converted frompeaks to banding patterns making areas of differential expression morereadily visible. Particular differentially expressed peaks/bands in thepositive cell line could be focused for better resolution and analysis.Overlaying the positive and negative plots obtained in each experimentshowed that the over-expression of proteins was seen only in thepositive cell lines (HepG2 and MCF-7) and these fractions were used forpeptide extraction purposes.

Peptide Extraction from Peak Fractions

Tryptic digestions were performed with sequencing grade trypsin in a20-hour peptide extraction process finally resulting in the extractionof peptides that were analyzed on a QSTAR Pulsar-I (ESI-qTOE-MS/MS),equipped with a nanosource with a working flow rate of 20-50 nL/min. Thepeptides ionize and are detected as doubly, triply or quadruply chargedmolecules which are then refined to their respective masses. De-novosequencing of the identified proteins was also performed wheneverpossible. Peptides were extracted from both positive and negative celllines to ensure it was the right antigen. Peptide masses extracted fromthe mass spectra were used directly to identify the antigen according tothe MOWSE scores obtained on protein databases that are accessiblethrough the MASCOT search engine.

Peptides were extracted post-tryptic digestion from the peak, fractionseluting at 15-18 minutes, from all four samples (MCF-7, HepG2, Panc-1and C-33A) and subjected them to MS analysis. In addition to fractionseluting at 15, 18 minutes, fractions eluting at the 12^(th) minute frompositive and negative cell lines were also processed simultaneously.FIGS. 11-14 show results of the TOF-MS scans of the peptides obtainedfrom the cell lines. As seen in FIG. 15, one single protein wasidentified corresponding to glucose transporter-8 from both the positivecell lines that was undetectable in the negative cell lines. Thedifference in elution between the two peaks (15 vs 18 minutes) could beattributed to changes in glycosylation or other post-translationalmodifications.

Mass Spectral Analysis

Peptide analysis was done in two ways:

-   -   All the peptides recovered and reconstructed to their right        masses were used directly in a peptide mass fingerprinting step        to obtain an ID for the protein.    -   Peptides that were abundant and well ionized were chosen for        further MS/MS ion fragmentation, wherein, the ‘y’ and ‘b’ ions        were used to deduce their primary structure. These sequences        were then searched for homologies in the protein database for        protein ID.

Peptides ionize and are detected as doubly, triply or quadruply chargedmolecules, on a LC-MS/MS system as opposed to detection as singlycharged on Matrix assisted ionization such as in MALDI. Differentiallycharged peptides were thereafter refined to their respective masses, inthe mass reconstruction step. These peptide masses were then directlyanalyzed by a matrix science based mascot search engine for antigen ID.Peptide masses extracted from the mass spectra were used directly toidentify the antigen according to the MOWSE scores obtained on proteindatabases that are accessible through search engines such as MASCOT,SEQUEST, and Prospector. Since the QSTAR-pulsar-I purchase includes thepurchase of license from Pepsea server for most recent protein databaseadditions, and is compatible with MASCOT, this search engine wasselected for all protein searches.

The list of peptides recovered and their mapped positions to thesequence from Glucose Transporter 8 are as given in FIGS. 15, 16 andTable 8. All peptides represented were obtained by de novo sequencing.FIG. 17 identifies Glucose Transporter 8 as the antigen.

MS/MS fragmentation of four of the peptides (1401.54-466.600000, 3+;1070.785448-536.400000, 2+; 1998.272862-667.098230, 3+;1176.185448-589.100000, 2+) gave rise to the fragment ions shown inFIGS. 18-21 that mapped to peptides from Glucose Transporter 8. Sincethese 2 peptides were all detected in TOF-MS, these peptides were usedfor MS/MS ion fragmentation apart from the peptides derived from massfingerprinting. A discrete nanospray head installed on a nanosource wasused for the purpose. The collision energy was 48V, curtain gas and CADgas were maintained at 25 and 6, respectively, and the sample allowed tocycle for 1.667 minutes (100 cycles) to obtain stable mass ionfragmentation. Peptides derived from the spectra clearly matched thesequence on Glucose Transporter 8, therefore were pulled down as majorhits. The ion fragmentation data further confirm the identity of GlucoseTransporter 8 as the cognate antigen for VB1-050.

Peptide mass fingerprinting and MS/MS fragmentation of theantigen-positive fractions revealed the identity of Glucosetransporter-8/GLUTX1/SLC 2A8 gene product as the cognate binding antigenfor VB1-050. Glucose transporter-8 is a ˜50 kDa type-II transmembraneprotein, with N-terminus inside the cell. 34% sequence coverage wasobtained from the peptides that were recovered in-house. Cell linesselected positive by flow show the presence of the antigen uponimmunoprecipitation. MS/MS analysis of two peptides, 1070.785, appearingas a doubly charged molecule (536.40000, 2+); 1401.54, appearing as atriply charged molecule (466.60000, 3+), identified two peptidesequences, SLASVVVGVIQ (SEQ ID NO:20 (292-303) and KTLEQITAHFEGR (SEQ IDNO:19) (466-477), respectively, clearly matched the protein sequencecorresponding to Glucose transporter-8.

MS/MS sequencing of two additional peptides recovered from MCF-7,1176.3547 and 1997.9992, mapped sequences with 68.2% homology tocorresponding peptides from GLUT8 with changes in amino acids at sevenpositions, i.e., 7, 10, 12-15, 18. The changes incorporated correspondto the positional changes at 12, 13 from LL to AA as reported by Shin etal. (2004, J. Neuro. Res. 75: 835), that is responsible for theorientation of GLUT8 from cytosol to the plasma membrane.

While the present invention has been described with reference to whatare presently considered to be the preferred examples, it is to beunderstood that the invention is not limited to the disclosed examples.To the contrary, the invention is intended to cover variousmodifications and equivalent arrangements included within the spirit andscope of the appended claims.

All publications, patents and patent applications are hereinincorporated by reference in their entirety to the same extent as ifeach individual publication, patent or patent application wasspecifically and individually indicated to be incorporated by referencein its entirety.

TABLE 1 CDR Sequences CDR Sequences VB1-050 L-chain H-chain CDR1RASQDISNYLA SEQ ID NO: 1 NYAMS SEQ ID NO: 4 CDR2 AASSLHS SEQ ID NO: 2AITPSGGSTNYADSVKG SEQ ID NO: 5 CDR3 LQYSTYPIT SEQ ID NO: 3 VPYRSTWYPLYSEQ ID NO: 6

TABLE 2 Comparison of tumor and normal cell surface reactivity withVB1-050 Clinical Representative Tumor Indication Cell lines N¹ MF²Relative Rank Breast MCF-7^(c), MDA-MB-231^(d), MDA-MB-435S^(a) 3 29.9 1Melanoma A-375, SK-MEL-5^(a,b), SK-MEL-28^(a) 3 22.7 2 OvarianSK-OV-3^(a), OVCar-3 2 21.7 3 Prostate DU-145^(a,b,f), PC-3^(a,b,g),LNCaP^(a,b,g) 3 19.6 4 Kidney Caki-1^(a), A498^(a), ACHN^(a) 3 18.4 5Rectum Sw837, NCI-H630 2 15.2 6 Lung A-549, NCI-H460, NCI-H69 3 14.8 7Liver SK-HEP-1, Hep-G2 2 14.6 8 Colon HT-29^(a), SW480, WiDr 3 13.3 9Cervix HeLa, C-41, C-33A 3 11.8 10 Head & Neck SCC-15, SCC-25 2 11.4 11Bladder UM-UC-3, T24 2 9.8 12 Stomach AGS, NCI-N-87, KATO III 3 9.6 13Pancreas PANC-1, BxPC-3, MIA PaCa-2 3 7.6 14 Endometrium RL-95-2,HEC-1-A 2 7.0 15 Normal Cell Type Cell Line Tumor normal Kidney HRE 112.5 1.5 Lung NHLF 1 8.7 1.7 Endothelial HUVEC 1 5.0 N/A Breast HMEC 13.1 9.6 Prostate PrEC 1 2.1 9.3 ¹N indicates the number of cell linestested per indication. ²MF: Values indicate the mean calculated from thesum of the mean fold increase in median fluorescence over the controlantibody from all cell lines in each indication. A zero value indicatesno measurable reactivity relative to the control antibody. ^(a)Indicatesorthotopic models offered by AntiCancer Inc. ^(b)Indicates cell linesavailable as GFP (green fluorescent protein)-transfectants.^(c)Her2/neu⁻, ER⁺. ^(d)Her2/neu, ER⁻, p53^(wt), ras^(wt).^(e)Her2/neu⁻, ER⁻, p53^(mt), ras^(wt). ^(f)Androgen-responsive.^(g)Androgen-unresponsive.

TABLE 3 LD Array of Formalin-Fixed Critical Normal Tissue for VB1-050Membrane Tissue Staining Score Range¹ Brain None (0/6) 0 Colon² None(0/4) 0 Heart None (0/5) 0 Kidney None (0/3) 0 Liver None (0/5) 0 LungNone (0/4) 0 Pancreas None (0/4) 0 Stomach³ None (0/4) 0 ¹Scoring wasevaluated on a 0-3+ scale, with 0 = no staining and trace being lessthan 1+ but greater than 0. Grades 1+ to 3+ represent increasedintensity of staining, with 3+ being strong, dark brown staining. Ingeneral, a single specimen of 6 different patients was screened. Wherefewer than 6 patients were screened indicates cores were either missingor were not representative of the tissue to be stained. Values inparentheses indicate the percentage of cells stained in the scoredrange. ²Only adjacent normal tissues were used. ³Four of five wereadjacent normal tissue specimens.

TABLE 4 HD Formalin-Fixed Normal TMA for VB1-050 Membrane TissueStaining Score Range* Adrenal None (0/5) 0 Aorta None (0/5) 0 ArteryNone (0/5) 0 Bladder None (0/5) 0 Brain None (0/5) 0 Breast None (0/5) 0Fallopian tube None (0/5) 0 LN None (0/4) 0 Muscle None (0/5) 0 OvaryNone (0/5) 0 Pituitary None (0/5) 0 Placenta None (0/5) 0 Prostate 0/5 0Skin 0/1 Spinal cord None (0/3) 0 Spleen None (0/5) 0 Testis 1/5 1+(30%) Thymus None (0/1) 0 Thyroid None (0/5) 0 Ureter 0/2 0 Uterus None(0/3) 0 *Scoring was evaluated on a 0-3+ scale, with 0 = no staining andtrace being less than 1+ but greater than 0. Grades 1+ to 3+ representincreased intensity of staining, with 3+ being strong, dark brownstaining. In general, 2 specimens of 8 different patients were screened.Where fewer than 8 patients were screened indicates cores were eithermissing or were not representative of the tissue to be stained. Valuesin parentheses indicate the percentage of cells stained in the scoredrange.

TABLE 5 HD Formalin-Fixed Tumor TMA for VB1-050 Membrane Tissue StainingScore Range Bladder 2/8 2+ (50%) Breast 1/8 1+ (90%) Cervix 1/8 1+ (30%)Colon 4/7   1+ (70-90%) Kidney 1/8 2+ (40%) Liver 3/6 1+ (80%) Lung 0/6N/A Ovary 3/7 1+ (20%) Pancreas 2/8   2+ (30-80%) Prostate 4/7   1+(20-60%) Rectum 0/7 N/A Skin 0/4 N/A Stomach 4/8 2+ (30%) Uterus 0/8 N/AHead & Neck 2/8   2+ (30-50%) Scoring was evaluated on a 0-3+ scale,with 0 = no staining and trace being less than 1+ but greater than 0.Grades 1+ to 3+ represent increased intensity of staining, with 3+ beingstrong, dark brown staining. In general, 2 specimens of 8 differentpatients were screened. Where fewer than 8 patients were screenedindicates cores were either missing or were not representative of thetissue to be stained. Head & neck cancers included carcinomas of thethroat, lip, larynx, mouth, tonsil, and gingival surface. Values inparentheses indicate the percentage of cells stained in the scoredrange. Cancer indications that are bolded indicate VB1-050 reactivity.

TABLE 6 Flow cytometry assessment of antibody binding as a function oftime and temperature Median Incubation Fluorescence Fold-increase %Reduction MAb ID Antibodies (min) at 37° C. (MF) in MF in MF VB1-050VB1-050 —⁴ 1041.0 ± 23.0  129.1 —  60 397.5 ± 5.1  49.2 61.8 120 317.5 ±4.1  39.3 69.6 Non- MA-103 — 536.1 ± 31.3 112.8 — Internalizing 120535.5 ± 16.8 113.0 — Control Internalizing 5E9 —⁴ 246 ± 11 60.0 —Control  60 53.5 ± 1.5 13.0 78.3 120 48 ± 4 11.7 80.5 ¹A representativeexperiment is shown. ²MF increase above the negative control, mousemyeloma IgG or human IgG (4B5). ³Percent reduction of MF from thecell-surface of tumor cells. ⁴(—) cells incubated on ice for 120minutes.

TABLE 7 Biological characterization of VB6-050 VB6 Saturation IgGconcentration Affinity (M) conc. (μg/mL) (μg/mL)* IC₅₀ (nM) VB6-0081.4.10⁻⁸ ND ND 280 ND: not determined *Concentration of IgG thatinhabits 50% of the VB6 binding.

TABLE 8 List of recovered peptides SEQ Observed Start End Peptide ID NO1998.27   3   22 PEDPSETEPAAPRPGASAPR 12 1151.241   6  15 PSETEPAAPR 133140.68  26  56 RVFLAAFAAALGPLSFGFALGYSSPA 14 IPSLQRA 2916.29  64  93RLDDAAASWFGAVVTLGAAAGGVLGG 15 WLVDRA 889.04 216 223 RQEAMAALRF 162984.32 224 249 RFLWGSEQGWEDPPIGAEQSFHLALL 17 RQ 4263.10 427 463KEFSSLMEVLRPYGAFWLASAFCIFS 18 VLFTLFCVPEIKG 1401.54 466 477KTLEQITAHFEGR 19 292 302 SLASVVVGVIQ 20

1. A cancer-associated variant of glucose transporter 8, comprising theamino acid sequence of SEQ ID NO:11.
 2. A cancer-associated variant ofglucose transporter 8, comprising the amino acid sequence of SEQ IDNO:12.
 3. A cancer-associated variant of glucose transporter 8,comprising the amino acid sequence of SEQ ID NO:13.
 4. Acancer-associated variant of glucose transporter 8, consisting of theamino acid sequence of SEQ ID NO:11.
 5. A pharmaceutical compositioncomprising an effective amount of a cancer-associated variant of glucosetransporter 8 comprising the amino acid sequence of SEQ ID NO:11, 12 or13, in admixture with a suitable diluent or carrier.
 6. Thepharmaceutical composition of claim 5, further comprising an adjuvant.7. A kit comprising the pharmaceutical composition according to claim 5and instructions for use thereof.